Method for multiplex detection of target nucleic acid based on CRISPR technology

Abstract

Based on the temperature tolerance of different Cas proteins, the present invention provides a method for multiplex detection of target nucleic acids based on CRISPR technology. Specifically, a method, a system and a kit for detecting target nucleic acids are provided. The detection method includes adding any one, any two, any three or all four of a first nucleic acid detection composition, a second nucleic acid detection composition, a third nucleic acid detection composition and a fourth nucleic acid detection composition to a reaction system containing target nucleic acids, and based on the temperature tolerance of different Cas proteins, multiplex detection of target nucleic acids can be achieved.

Description

[0001] This application is a divisional application of the invention patent application with the application date of September 18, 2020, application number 202010987874.6, and invention title "Method for multiplex detection of target nucleic acid based on CRISPR technology". Technical Field

[0002] The present invention relates to the field of nucleic acid detection, and relates to a method for multiplex detection of target nucleic acid based on CRISPR technology. Specifically, it relates to a method, system and kit for detecting target nucleic acid based on CRISPR technology, and particularly relates to a method for multiplex detection of target nucleic acid based on CRISPR technology. Background Art

[0003] Specific methods for detecting nucleic acid molecules have important application values, such as the detection of pathogens and genetic diseases. In terms of pathogen detection, since each pathogen microorganism has its unique characteristic nucleic acid molecule sequence, nucleic acid molecule detection specific to a particular species can be developed, also known as nucleic acid diagnostics (NADs), which is of great significance in the fields of food safety, detection of environmental microbial pollution, and human pathogen infection. Another aspect is the detection of single nucleotide polymorphisms (SNPs) in humans or other species. Understanding the relationship between genetic variation and biological function at the genomic level provides a new perspective for modern molecular biology. Among them, SNPs are closely related to biological functions, evolution, and diseases, so the development of detection and analysis technologies for SNPs is particularly important.

[0004] Currently, the established specific nucleic acid molecule detection generally needs to be divided into two steps. The first step is the amplification of the target nucleic acid, and the second step is the detection of the target nucleic acid. Existing detection technologies include methods such as restriction endonuclease method, Southern, Northern, dot blot, fluorescence PCR detection technology, LAMP loop-mediated isothermal amplification technology, recombinase polymerase amplification technology (RPA), etc. After 2012, the CRISPR gene editing technology emerged. The Zhang Feng team developed a new nucleic acid diagnostic technology (SHERLOCK technology) targeting RNA with Cas13 as the core based on the RPA technology. The Doudna team developed a diagnostic technology (DETECTR technology) with Cas12 enzyme as the core. Wang Jin, a doctor from the Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, also developed a new nucleic acid detection technology (HOLMES technology) based on Cas12. Nucleic acid detection technologies developed based on CRISPR technology are playing an increasingly important role.

[0005] Although there are numerous existing nucleic acid detection technologies, how to detect more quickly, simply, inexpensively, and accurately remains an important direction for improving detection technologies. In particular, how to perform multiplex detection of nucleic acids is an urgent problem to be solved. Summary of the Invention

[0006] Based on the differences in the highest temperatures tolerated by different Cas proteins, the present invention can achieve multiplex detection of target nucleic acids by setting different reaction temperatures. The present invention provides a method for nucleic acid detection based on CRISPR technology, especially a method, system, and kit for multiplex detection of nucleic acids.

[0007] On the one hand, the present invention provides a method for detecting target nucleic acids in a sample, the method comprising contacting the sample with a nucleic acid detection composition and a single-stranded nucleic acid detector, the nucleic acid detection composition comprising a Cas protein and a gRNA; the gRNA comprising a region that binds to the Cas protein and a guide sequence that hybridizes to a target sequence on the target nucleic acid;

[0008] The method comprises subjecting the sample to any one, any two, any three, or all four of the following reactions I-IV:

[0009] I. Contacting the sample with a first nucleic acid detection composition and a single-stranded nucleic acid detector and reacting for a first period of time at a first temperature;

[0010] II. Contacting the sample with a second nucleic acid detection composition and a single-stranded nucleic acid detector and reacting for a second period of time at a second temperature;

[0011] III. Contacting the sample with a third nucleic acid detection composition and a single-stranded nucleic acid detector and reacting for a third period of time at a third temperature;

[0012] IV. Contacting the sample with a fourth nucleic acid detection composition and a single-stranded nucleic acid detector and reacting for a fourth period of time at a fourth temperature;

[0013] The first nucleic acid detection composition comprises Cas12i, a first gRNA that can bind to Cas12i and hybridize to a first target sequence on the target nucleic acid;

[0014] The second nucleic acid detection composition comprises Cas12a, a second gRNA that can bind to Cas12a and hybridize to a second target sequence on the target nucleic acid;

[0015] The third nucleic acid detection composition comprises Cas12j, a third gRNA that can bind to Cas12j and hybridize to a third target sequence on the target nucleic acid;

[0016] The fourth nucleic acid detection composition includes Cas12b, a fourth gRNA that can bind to Cas12b and hybridize with a fourth target sequence on the target nucleic acid;

[0017] The first temperature is 4°C - 50°C, preferably 20°C - 50°C, more preferably 37°C - 50°C, for example, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C;

[0018] The second temperature is 4°C - 48°C, preferably 20°C - 48°C, more preferably 37°C - 48°C, for example, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C or 48°C;

[0019] The third temperature is 4°C - 56°C, preferably 20°C - 56°C, more preferably 37°C - 56°C, even more preferably 48°C - 56°C, for example, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C;

[0020] The fourth temperature is 4°C - 80°C, preferably 20°C - 75°C, more preferably 37°C - 70°C, even more preferably 48°C - 70°C, for example, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C;

[0021] Detect the detectable signal generated by the Cas protein cleavage of the single-stranded nucleic acid detector, thereby detecting the target nucleic acid.

[0022] In one embodiment, the above single-stranded nucleic acid detector is a single-stranded nucleic acid detector. This setting makes the entire detection method more convenient and fast.

[0023] In other embodiments, the above single-stranded nucleic acid detector can also be a combination of multiple single-stranded nucleic acid detectors. The multiple single-stranded nucleic acid detectors can be universal for different Cas proteins (i.e., any Cas protein can cleave the above single-stranded nucleic acid detector); the multiple single-stranded nucleic acid detectors can also include single-stranded nucleic acid detections specific for a certain Cas protein or certain Cas proteins, but it is necessary to ensure that each Cas protein has at least one single-stranded nucleic acid detector that can be cleaved.

[0024] Furthermore, the sample can be a single sample or a mixed sample.

[0025] In one embodiment, any two of the above reactions are selected from any of the following combinations:

[0026] (1) I and III, where the third temperature is higher than the first temperature; preferably, the first temperature is 37°C - 48°C and the third temperature is 50°C - 56°C;

[0027] (2) I and IV, where the fourth temperature is higher than the first temperature; preferably, the first temperature is 37°C - 48°C and the fourth temperature is 50°C - 70°C;

[0028] (3) III and IV, where the fourth temperature is higher than the third temperature; preferably, the third temperature is 50°C - 56°C and the fourth temperature is 58°C - 70°C;

[0029] (4) II and III, where the third temperature is higher than the second temperature; preferably, the second temperature is 37°C - 48°C and the third temperature is 50°C - 56°C;

[0030] (5) II and IV, where the fourth temperature is higher than the second temperature; preferably, the second temperature is 37°C - 48°C and the fourth temperature is 50°C - 70°C.

[0031] In other embodiments, the reactions of any three of the above are selected from any of the following combinations:

[0032] (1) I, III and IV, where the fourth temperature is higher than the third temperature and the third temperature is higher than the first temperature; preferably, the first temperature is 37°C - 48°C, the third temperature is 50°C - 56°C, and the fourth temperature is 58°C - 70°C;

[0033] (2) II, III and IV; where the fourth temperature is higher than the third temperature and the third temperature is higher than the second temperature; preferably, the second temperature is 37°C - 48°C, the third temperature is 50°C - 56°C, and the fourth temperature is 58°C - 70°C.

[0034] The combinations of the above different reaction conditions can be to contact the sample with different nucleic acid detection compositions simultaneously, react at a certain temperature for a period of time first, and then react at other temperatures for a period of time; or it can be to contact the sample with a certain detection composition first, react at the corresponding reaction temperature for a period of time, and then, contact with other detection compositions and react at the corresponding reaction temperature for another period of time.

[0035] Taking the combination of the reaction conditions of I and III as an example, the sample can be contacted with the first detection composition and the third detection composition simultaneously, and then react at the first temperature for a period of time, and then react at the third temperature for a period of time; or, the sample can be contacted with the first detection composition first, react at the first temperature for a period of time, and then, contact with the third detection composition and react at the third temperature for a period of time.

[0036] On the other hand, the present invention provides a method for multiplex detection of target nucleic acids in a sample, the method comprising contacting the sample with a nucleic acid detection composition and a single-stranded nucleic acid detector under any one of the following conditions i-vii, the nucleic acid detection composition comprising a Cas protein and a gRNA; the gRNA comprising a region that binds to the Cas protein and a guide sequence that hybridizes to a target sequence on the target nucleic acid; detecting a detectable signal generated by the Cas protein cleaving the single-stranded nucleic acid detector, thereby detecting the target nucleic acid; the conditions of i-vii are as follows:

[0037] i. Contact the sample with the above-mentioned first nucleic acid detection composition and the third nucleic acid detection composition, react for a first period of time at the above-mentioned first temperature, and then react for a third period of time at the third temperature; the third temperature is higher than the first temperature; preferably, the first temperature is 37°C - 48°C, and the third temperature is 50°C - 56°C;

[0038] ii. Contact the sample with the above-mentioned first nucleic acid detection composition and the fourth nucleic acid detection composition, react for a first period of time at the above-mentioned first temperature, and then react for a fourth period of time at the fourth temperature; the fourth temperature is higher than the first temperature; preferably, the first temperature is 37°C - 48°C, and the fourth temperature is 50°C - 70°C;

[0039] iii. Contact the sample with the above-mentioned third nucleic acid detection composition and the fourth nucleic acid detection composition, react for a third period of time at the above-mentioned third temperature, and then react for a fourth period of time at the fourth temperature; the fourth temperature is higher than the third temperature; preferably, the third temperature is 50°C - 56°C, and the fourth temperature is 58°C - 70°C;

[0040] iv. Contact the sample with the above-mentioned second nucleic acid detection composition and the third nucleic acid detection composition, react for a second period of time at the above-mentioned second temperature, and then react for a third period of time at the third temperature; the third temperature is higher than the second temperature; preferably, the second temperature is 37°C - 48°C, and the third temperature is 50°C - 56°C;

[0041] v. Contact the sample with the above-mentioned second nucleic acid detection composition and the fourth nucleic acid detection composition, react for a second period of time at the above-mentioned second temperature, and then react for a fourth period of time at the fourth temperature; the fourth temperature is higher than the second temperature; preferably, the second temperature is 37°C - 48°C, and the fourth temperature is 50°C - 70°C;

[0042] vi. Contact the sample with the above-mentioned first nucleic acid detection composition, third nucleic acid detection composition, and fourth nucleic acid detection composition, react at a first temperature for a first period of time, then react at a third temperature for a third period of time, and then react at a fourth temperature for a fourth period of time; the fourth temperature is higher than the third temperature, and the third temperature is higher than the first temperature; preferably, the first temperature is 37°C - 48°C, the third temperature is 50°C - 56°C, and the fourth temperature is 58°C - 70°C;

[0043] vii. Contact the sample with the above-mentioned second nucleic acid detection composition, third nucleic acid detection composition, and fourth nucleic acid detection composition, react at a second temperature for a second period of time, then react at a third temperature for a third period of time, and then react at a fourth temperature for a fourth period of time; the fourth temperature is higher than the third temperature, and the third temperature is higher than the second temperature; preferably, the second temperature is 37°C - 48°C, the third temperature is 50°C - 56°C, and the fourth temperature is 58°C - 70°C.

[0044] In the present invention, the detectable signal is achieved by the following methods: visual-based detection, sensor-based detection, color detection, fluorescence signal-based detection, gold nanoparticle-based detection, fluorescence polarization, colloid phase transition / dispersion, electrochemical detection, and semiconductor-based detection.

[0045] In the present invention, the detectable signal can be any signal generated when the single-stranded nucleic acid detector is cleaved. For example, gold nanoparticle-based detection, fluorescence polarization, colloid phase transition / dispersion, electrochemical detection, semiconductor-based sensing. The detectable signal can be read out by any suitable means, including but not limited to: measurement of detectable fluorescence signals, gel electrophoresis detection (by detecting changes in bands on the gel), detection of the presence or absence of color based on vision or sensors, or differences in the presence of color (e.g., based on gold nanoparticles) and differences in electrical signals.

[0046] Preferably, a fluorescent group and a quenching group are respectively provided at both ends of the single-stranded nucleic acid detector. When the single-stranded nucleic acid detector is cleaved, a detectable fluorescence signal can be exhibited. The fluorescent group is selected from one or any combination of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red, or LC RED460; the quenching group is selected from one or any combination of BHQ1, BHQ2, BHQ3, Dabcy1, or Tamra.

[0047] In other embodiments, different labeling molecules are respectively provided at the 5'-end and 3'-end of the single-stranded nucleic acid detector. By means of colloidal gold detection, the colloidal gold test results before and after the single-stranded nucleic acid detector is cleaved by the Cas protein are detected; different color development results will be shown on the detection line and quality control line of colloidal gold before and after the single-stranded nucleic acid detector is cleaved by the Cas protein.

[0048] In the present invention, the first target sequence, the second target sequence, the third target sequence, and the fourth target sequence may be the same target sequence, or may be different target sequences from each other.

[0049] Those skilled in the art can select according to actual needs whether the above-mentioned first target sequence, second target sequence, third target sequence, and fourth target sequence are the same, or different, or partially the same.

[0050] Preferably, the above-mentioned target sequences are different target sequences from each other. With such an arrangement, the method for detecting target nucleic acid of the present invention can achieve multiplex detection of nucleic acids in a sample; in one embodiment, the first target sequence, the second target sequence, the third target sequence, and the fourth target sequence may be target sequences designed for different sites of the same target nucleic acid or the same gene, or may be target sequences designed for different target nucleic acids or different genes. In one embodiment, different target sequences can be designed for a certain type of bacteria, virus or disease-related nucleic acid; in other embodiments, different target sequences can be designed for different types of bacteria, viruses or disease-related nucleic acids.

[0051] For example, when performing dual detection using the first nucleic acid detection composition and the second nucleic acid detection composition, different target sequences can be designed for the SARS-CoV2 (COVID-19) virus to perform dual detection on two target nucleic acids of SARS-CoV2 (COVID-19); or, the first target sequence and the second target sequence can be designed for SARS-CoV2 (COVID-19) and SARS viruses respectively, so as to perform dual detection on SARS-CoV2 (COVID-19) and SARS viruses.

[0052] The durations of the first time period, the second time period, the third time period, and the fourth time period are 2-30 minutes, preferably 3-20 minutes, more preferably 4-10 minutes, and even more preferably 5 minutes.

[0053] On the other hand, the present invention also provides a system for detecting a target nucleic acid in a sample, the system comprising a nucleic acid detection composition, the nucleic acid detection composition comprising a Cas protein, a gRNA, and a single-stranded nucleic acid detector; the gRNA comprising a region that binds to the Cas protein and a guide sequence that hybridizes to a target sequence on the target nucleic acid; the nucleic acid detection composition being selected from any one, any two, any three, or all four of the above-mentioned first nucleic acid detection composition, second nucleic acid detection composition, third nucleic acid detection composition, and fourth nucleic acid detection composition.

[0054] On the other hand, the present invention also provides a kit for detecting a target nucleic acid in a sample, the kit comprising a nucleic acid detection composition, the nucleic acid detection composition comprising a Cas protein, a gRNA, and a single-stranded nucleic acid detector; the gRNA comprising a region that binds to the Cas protein and a guide sequence that hybridizes to a target sequence on the target nucleic acid. The nucleic acid detection composition is selected from any one, any two, any three, or all four of the above-mentioned first nucleic acid detection composition, second nucleic acid detection composition, third nucleic acid detection composition, and fourth nucleic acid detection composition.

[0055] On the other hand, the present invention also provides the use of the above system or kit in detecting a target nucleic acid in a sample. As described above, when detecting a target nucleic acid in a sample, the system or kit of the present invention can use one or any combination of the first nucleic acid detection composition, second nucleic acid detection composition, third nucleic acid detection composition, and fourth nucleic acid detection composition to detect the same target sequence, or detect different target sequences, thereby achieving a dual, triple, or quadruple detection effect.

[0056] On the other hand, the present invention also provides the use of a nucleic acid detection composition in preparing a detection kit for detecting a target nucleic acid in a sample; the nucleic acid detection composition is selected from one or any combination of the above-mentioned first nucleic acid detection composition, second nucleic acid detection composition, third nucleic acid detection composition, and fourth nucleic acid detection composition; the first nucleic acid detection composition is contacted with the sample at the first temperature, the second nucleic acid detection composition is contacted with the sample at the second temperature, the third nucleic acid detection composition is contacted with the sample at the third temperature, and the fourth nucleic acid detection composition is contacted with the sample at the fourth temperature.

[0057] In a preferred embodiment, the detection kit is a kit suitable for multiplex detection of a sample at different temperatures; the kit comprises any two, three, or four of the above-mentioned first nucleic acid detection composition, second nucleic acid detection composition, third nucleic acid detection composition, and fourth nucleic acid detection composition.

[0058] Furthermore, the kit further comprises a single-stranded nucleic acid detector.

[0059] Furthermore, the kit is also provided with a positive control.

[0060] In the present invention, the single-stranded nucleic acid detector includes, but is not limited to, single-stranded DNA, single-stranded RNA, DNA-RNA hybrids, nucleic acid analogs, base modifiers, and single-stranded nucleic acid detectors containing abasic spacers, etc.; "nucleic acid analogs" include, but are not limited to: locked nucleic acid, bridged nucleic acid, morpholino nucleic acid, ethylene glycol nucleic acid, hexitol nucleic acid, threose nucleic acid, arabinose nucleic acid, 2'-O-methyl RNA, 2'-methoxyacetyl RNA, 2'-fluoro RNA, 2'-amino RNA, 4'-thio RNA, and combinations thereof, including optionally ribonucleotide or deoxyribonucleotide residues.

[0061] In the present invention, the target nucleic acid includes ribonucleotides or deoxyribonucleotides, including single-stranded nucleic acids and double-stranded nucleic acids, such as single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA.

[0062] In some embodiments, the method of the present invention further includes the step of measuring a detectable signal generated by a CRISPR / CAS effector protein (Cas protein). The Cas protein can recognize the target nucleic acid or hybridize with the target nucleic acid and then activate the cleavage activity of the single-stranded nucleic acid, thereby cleaving the single-stranded nucleic acid detector and generating a detectable signal.

[0063] In one embodiment, the target nucleic acid is derived from samples such as viruses, bacteria, microorganisms, soil, water sources, the human body, animals, plants, etc. Preferably, the target nucleic acid is a product enriched or amplified by methods such as PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM, etc.

[0064] In one embodiment, the method of the present invention further includes the step of obtaining the target nucleic acid from a sample.

[0065] In one embodiment, the target nucleic acid is viral nucleic acid, bacterial nucleic acid, specific nucleic acid related to a disease, such as a specific mutation site or SNP site or nucleic acid different from a control; preferably, the virus is a plant virus or an animal virus, for example, papillomavirus, hepadnavirus, herpesvirus, adenovirus, poxvirus, parvovirus, coronavirus; preferably, the virus is a coronavirus, preferably, SARS, SARS-CoV2 (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-Cov.

[0066] In some embodiments, the target nucleic acid is derived from cells, for example, from cell lysates.

[0067] In some embodiments, the measurement of the detectable signal can be quantitative, and in other embodiments, the measurement of the detectable signal can be qualitative.

[0068] In one embodiment, the method further includes the step of obtaining the target nucleic acid from the sample.

[0069] In some embodiments, the target nucleic acid is derived from cells, for example, from cell lysates.

[0070] In some embodiments, the measurement of the detectable signal can be quantitative, and in other embodiments, the measurement of the detectable signal can be qualitative.

[0071] In the present invention, the guiding sequence includes 10 - 40 bp; preferably, 12 - 25 bp; preferably, 15 - 23 bp; preferably, 16 - 18 bp.

[0072] In the present invention, the gRNA has at least 50% sequence identity with the target sequence on the target nucleic acid, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.

[0073] In one embodiment, when the target sequence contains one or more characteristic sites (such as specific mutation sites or SNPs), the characteristic sites are completely matched with the gRNA.

[0074] In one embodiment, the detection method may include one or more gRNAs with different guiding sequences that target different target sequences.

[0075] In one embodiment, the Cas12a is selected from one or any combination of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a; the Cas12a is preferably LbCas12a, and its amino acid sequence is as shown in SEQ ID No.1, or a derivative protein formed by substitution, deletion or addition of one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues of the amino acid sequence shown in SEQ ID No.1 and having substantially the same function.

[0076] In other embodiments, the amino acid sequence of Cas 12b is as shown in SEQ ID No.2, or a derivative protein having substantially the same function formed by substituting, deleting or adding one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues to the amino acid sequence shown in SEQ ID No.2 or its active fragment.

[0077] In other embodiments, the amino acid sequence of Cas 12i is as shown in SEQ ID No.3, or a derivative protein having substantially the same function formed by substituting, deleting or adding one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues to the amino acid sequence shown in SEQ ID No.3 or its active fragment.

[0078] In other embodiments, the amino acid sequence of Cas 12j is as shown in SEQ ID No.4, or a derivative protein having substantially the same function formed by substituting, deleting or adding one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues to the amino acid sequence shown in SEQ ID No.4 or its active fragment.

[0079] The terms "hybridize" or "complementary" or "substantially complementary" mean that a nucleic acid (e.g., RNA, DNA) contains a nucleotide sequence that enables it to non-covalently bind, i.e., to form base pairs and / or G / U base pairs with another nucleic acid in a sequence-specific, anti-parallel manner (i.e., the nucleic acid specifically binds to a complementary nucleic acid), "anneal" or "hybridize". Hybridization requires that the two nucleic acids contain complementary sequences, although there may be mismatches between the bases. The appropriate conditions for hybridization between two nucleic acids depend on the length and degree of complementarity of the nucleic acids, which are variables known in the art. Typically, the length of the hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

[0080] It should be understood that the sequence of a polynucleotide does not need to be 100% complementary to the sequence of its target nucleic acid to specifically hybridize. The polynucleotide may contain 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 98% or higher, 99% or higher, 99.5% or higher, or 100% complementarity to the sequence of the target region in the target nucleic acid sequence with which it hybridizes.

[0081] General definitions:

[0082] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0083] The term "amino acid" refers to a carboxylic acid containing an amino group. All kinds of proteins in organisms are composed of 20 basic amino acids.

[0084] The terms "polynucleotide", "nucleotide sequence", "nucleic acid sequence", "nucleic acid molecule" and "nucleic acid" are used interchangeably and include DNA, RNA, or their hybrids, and may be double-stranded or single-stranded.

[0085] The term "oligonucleotide" refers to a sequence containing 3 - 100 nucleotides, preferably 3 - 30 nucleotides, more preferably 4 - 20 nucleotides, and even more preferably 5 - 15 nucleotides.

[0086] The terms "homology" or "identity" are used to refer to the sequence match between two polypeptides or between two nucleic acids. When the same base or amino acid monomer subunit occupies a certain position in both of the two sequences being compared (for example, adenine occupies a certain position in each of two DNA molecules, or lysine occupies a certain position in each of two polypeptides), then the molecules are identical at that position. Between two sequences. Generally, the comparison is made when the two sequences are aligned to yield maximum identity. Such alignment can be performed by using, for example, the identity of amino acid sequences can be determined by conventional methods, referring to, for example, Smith and Waterman, 1981, Adv. Appl. Math. 2:482; Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444; Thompson et al., 1994, Nucleic Acids Res 22:467380, etc., through computerized algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group). The BLAST algorithm available from the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov / ) can also be used with default parameters to determine it.

[0087] As used herein, the term "CRISPR" refers to Clustered Regularly Interspaced Short Palindromic Repeats, which is from the immune system of microorganisms.

[0088] As used herein, "biotin" is also known as vitamin H, and is a small molecule vitamin with a molecular weight of 244 Da. "Avidin", also known as streptavidin, is an alkaline glycoprotein with four binding sites that have a very high affinity for biotin. The commonly used avidin is streptavidin. The extremely strong affinity between biotin and avidin can be used to amplify or enhance the detection signal in a detection system. For example, biotin can easily bind to proteins (such as antibodies) by covalent bonds, and the avidin molecule conjugated with an enzyme reacts with the biotin molecule conjugated with a specific antibody, which not only plays a multi-stage amplification role, but also produces color due to the catalytic action of the enzyme when encountering the corresponding substrate, so as to achieve the purpose of detecting unknown antigen (or antibody) molecules.

[0089] Target nucleic acid

[0090] As used herein, the "target nucleic acid" refers to a polynucleotide molecule extracted from a biological sample (sample to be tested). The biological sample is any solid or fluid sample obtained, excreted or secreted from any organism, including but not limited to single-celled organisms such as bacteria, yeast, protozoa and amoeba, etc., multi-cellular organisms (such as plants or animals, including samples from healthy or seemingly healthy human subjects or human patients affected by diseases or disorders to be diagnosed or investigated, such as infections by pathogenic microorganisms such as pathogenic bacteria or viruses). For example, the biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, feces, sputum, mucus, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any body secretion, exudate, effusion (for example, the fluid obtained from an abscess or any other infected or inflamed site), or a fluid obtained from a joint (such as a normal joint or a joint affected by a disease, such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis), or a swab of the skin or mucosal surface. The sample can also be a sample obtained from any organ or tissue (including biopsy or autopsy specimens, such as tumor biopsies) or can contain cells (primary cells or cultured cells) or a culture medium conditioned by any cell, tissue or organ. Exemplary samples include but are not limited to, cells, cell lysates, blood smears, cytocentrifuge preparations, cytology smears, body fluids (such as blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.), tissue biopsies (such as tumor biopsies), fine needle aspirates and / or tissue sections (such as cryostat tissue sections and / or paraffin-embedded tissue sections).

[0091] In other embodiments, the biological sample can be plant cells, callus, tissues or organs (such as roots, stems, leaves, flowers, seeds, fruits), etc.

[0092] In the present invention, the target nucleic acid further includes a DNA molecule formed by reverse transcription of RNA. Further, the target nucleic acid can be amplified by techniques well known in the art. The amplification techniques include isothermal amplification techniques and non-isothermal amplification techniques. Isothermal amplification can be nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), or nicking enzyme amplification reaction (NEAR). In certain exemplary embodiments, non-isothermal amplification methods can be used, including but not limited to PCR, multiple displacement amplification (MDA), rolling circle amplification (RCA), ligase chain reaction (LCR), or derivative amplification method (RAM).

[0093] Further, the detection method of the present invention further includes a step of amplifying the target nucleic acid; the detection system further includes reagents for amplifying the target nucleic acid. The amplification reagents include one or more of the following: DNA polymerase, strand displacement enzyme, helicase, recombinase, single-stranded binding protein, etc.

[0094] Cas protein

[0095] As used herein, "Cas protein" refers to CRISPR-associated protein, preferably from type V or type VI CRISPR / CAS protein. Once it binds to the target sequence to be detected (the target sequence) (i.e., forms a ternary complex of Cas protein-gRNA-target sequence), it can induce its trans activity, that is, randomly cleave non-target single-stranded nucleotides (i.e., the single-stranded nucleic acid detector described herein). When the Cas protein binds to the target sequence, whether it cleaves or does not cleave the target sequence, it can induce its trans activity; preferably, it induces its trans activity by cleaving the target sequence; more preferably, it induces its trans activity by cleaving the single-stranded target sequence. The Cas protein recognizes the target sequence by recognizing the PAM (protospacer adjacent motif) adjacent to the target sequence.

[0096] The Cas protein of the present invention is a protein having at least trans cleavage activity. Preferably, the Cas protein is a protein having both Cis and trans cleavage activities. The Cis activity refers to the activity of the Cas protein to recognize the PAM site and specifically cleave the target sequence under the action of gRNA.

[0097] The Cas proteins described in the present invention include type V CRISPR / CAS effector proteins, including protein families such as Cas 12 and Cas14. Preferably, for example, Cas12 proteins, such as Cas12a, Cas12b, Cas12i, Cas12j; preferably, the Cas protein is Cas12a, Cas12b, Cas12i, Cas12j; the Cas14 protein family includes Cas14a, Cas14b, etc.

[0098] In an embodiment, the Cas proteins referred to herein, such as Cas 12, also encompass functional variants or homologs or orthologs thereof. As used herein, a "functional variant" of a protein refers to a variant of such a protein that retains at least part of the activity of the protein. Functional variants can include mutants (which can be insertion, deletion or substitution mutants), including polymorphs, etc. Also included among functional variants are fusion products of such proteins with another nucleic acid, protein, polypeptide or peptide that is not normally related. Functional variants can be naturally occurring or can be man-made. Advantageous embodiments can relate to engineered or non-naturally occurring type V DNA-targeting effector proteins.

[0099] In one embodiment, one or more nucleic acid molecules encoding a Cas protein, such as Cas12, or its orthologs or homologs can be codon-optimized for expression in eukaryotic cells. Eukaryotes can be as described herein. The one or more nucleic acid molecules can be engineered or non-naturally occurring.

[0100] In one embodiment, the Cas12 protein or its orthologs or homologs can comprise one or more mutations (and thus the nucleic acid molecule encoding it can have one or more mutations. The mutations can be artificially introduced mutations and can include, but are not limited to, one or more mutations in the catalytic domain.

[0101] In one embodiment, the Cas protein can be from: Leptotrichia, Listeria, Corynebacterium, Sutterella, Legionella, Treponema, Lineola, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Azospirillum, Sphaerochaeta, Gluconacetobacter, Neisseria, Rothia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, Campylobacter, and Lachnospira.

[0102] In one embodiment, the Cas protein is selected from the group consisting of the following sequences:

[0103] (1) the proteins shown in SEQ ID No.1-4;

[0104] (2) A derivative protein formed by substitution, deletion or addition of one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues to the amino acid sequences shown in SEQ ID No. 1-4 or their active fragments, and having substantially the same function.

[0105] In one embodiment, the Cas protein further includes a protein having 50%, preferably 55%, preferably 60%, preferably 65%, preferably 70%, preferably 75%, preferably 80%, preferably 85%, preferably 90%, preferably 95% sequence identity with the above sequence and having trans activity.

[0106] The Cas protein can be obtained by recombinant expression vector technology, that is, a nucleic acid molecule encoding the protein is constructed onto a suitable vector and then transformed into a host cell, so that the encoding nucleic acid molecule is expressed in the cell to obtain the corresponding protein. The protein can be secreted by the cell or obtained by disrupting the cell through conventional extraction techniques. The encoding nucleic acid molecule can be integrated into the genome of the host cell for expression or can be expressed without integration into the host cell. The vector further includes regulatory elements that facilitate sequence integration or self-replication. The vector can be of types such as plasmids, viruses, cosmids, phages, etc., which are well known to those skilled in the art. Preferably, the expression vector in the present invention is a plasmid. The vector further includes one or more regulatory elements selected from promoters, enhancers, ribosome binding sites for translation initiation, terminators, polyadenylation sequences, and selection marker genes.

[0107] The host cell can be a prokaryotic cell, such as Escherichia coli, Streptomyces, Agrobacterium; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Those of ordinary skill in the art are well aware of how to select appropriate vectors and host cells.

[0108] gRNA

[0109] As used herein, the "gRNA" is also known as guide RNA or guiding RNA and has the meaning commonly understood by those skilled in the art. Generally speaking, the guiding RNA may comprise direct repeat sequences and a guiding sequence, or consist essentially of or consist of direct repeat sequences and a guiding sequence (also known as a spacer sequence in the context of an endogenous CRISPR system). In different CRISPR systems, depending on the Cas protein it relies on, the gRNA may include crRNA and tracrRNA, or may only contain crRNA. crRNA and tracrRNA can be artificially modified and fused to form single guide RNA (sgRNA). In some cases, the guiding sequence is any polynucleotide sequence that has sufficient complementarity with the target sequence (the characteristic sequence described in the present invention) to hybridize with the target sequence and guide the specific binding of the CRISPR / Cas complex to the target sequence, usually having a sequence length of 12 - 25 nt. The aforementioned direct repeat sequences can fold to form a specific structure (such as a stem-loop structure) for Cas protein recognition to form a complex. The guiding sequence does not need to be 100% complementary to the characteristic sequence (target sequence). The guiding sequence is not complementary to the single-stranded nucleic acid detector.

[0110] In certain embodiments, when optimally aligned, the degree of complementarity (match degree) between the guiding sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Determining the optimal alignment is within the capabilities of those of ordinary skill in the art. For example, there are publicly available and commercially available alignment algorithms and programs, such as but not limited to ClustalW, the Smith-Waterman algorithm in matlab, Bowtie, Geneious, Biopython, and SeqMan.

[0111] The gRNA described in the present invention can be natural, or can be artificially modified or designed and synthesized.

[0112] Single-stranded nucleic acid detector

[0113] Both ends of the single-stranded nucleic acid detector of the present invention include different reporter groups or labeling molecules, which do not exhibit a reporter signal when in its initial state (i.e., the uncut state), and when the single-stranded nucleic acid detector is cut, a detectable signal is presented, that is, there is a detectable difference after cutting compared to before cutting. In the present invention, if a detectable difference can be detected, it reflects that the target nucleic acid contains the characteristic sequence to be detected; or, if the aforementioned detectable difference cannot be detected, it reflects that the target nucleic acid does not contain the characteristic sequence to be detected.

[0114] In one embodiment, the reporting group or labeling molecule includes a fluorescent group and a quenching group. The fluorescent group is selected from one or any combination of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red, or LC RED460; and the quenching group is selected from one or any combination of BHQ1, BHQ2, BHQ3, Dabcy1, or Tamra.

[0115] In one embodiment, the single-stranded nucleic acid detector has a first molecule (such as FAM or FITC) connected to the 5'-end and a second molecule (such as biotin) connected to the 3'-end. The reaction system containing the single-stranded nucleic acid detector is used in combination with a flow strip to detect a characteristic sequence (preferably, in a colloidal gold detection mode). The flow strip is designed to have two capture lines. An antibody that binds to the first molecule (i.e., the first molecule antibody) is provided at the sample contact end (colloidal gold). An antibody that binds to the first molecule antibody is contained at the first line (control line), and an antibody of the second molecule that binds to the second molecule (i.e., the second molecule antibody, such as avidin) is contained at the second line (test line). When the reaction flows along the strip, the first molecule antibody binds to the first molecule and carries the cleaved or uncleaved oligonucleotide to the capture lines. The cleaved reporter will bind to the antibody of the first molecule antibody at the first capture line, and the uncleaved reporter will bind to the second molecule antibody at the second capture line. The binding of the reporting group to each line will result in a strong readout / signal (such as color). As more reporters are cleaved, more signals will accumulate at the first capture line, and fewer signals will appear at the second line. In some aspects, the present invention relates to the use of a flow strip as described herein for detecting nucleic acids. In some aspects, the present invention relates to a method for detecting nucleic acids using a flow strip defined herein, such as a (lateral) flow test or a (lateral) flow immunochromatographic assay. In some aspects, the molecules in the single-stranded nucleic acid detector can be mutually replaced, or the positions of the molecules can be changed, as long as the reporting principle is the same as or similar to that of the present invention, and the improved methods are also included in the present invention. Description of the Drawings

[0116] Figure 1. Temperature tolerance results when Cas12i performs nucleic acid detection.

[0117] Figure 2. Temperature tolerance results when Cas12j performs nucleic acid detection.

[0118] Figure 3. Temperature tolerance results when Cas12b performs nucleic acid detection.

[0119] Figure 4 . LAMP amplification results for the OsTGW6 gene and the EV71 gene.

[0120] Figure 5 .Utilize the dual detection results of Cas12i and Cas12j.

[0121] Figure 6 .Utilize the triple detection results of Cas12i, Cas12j and Cas12b. Embodiment

[0122] The following further illustrates the present invention in conjunction with embodiments. The following description is only for the preferred embodiments of the present invention, and does not limit the present invention in other forms. Any person skilled in the art may use the disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention's solution falls within the protection scope of the present invention.

[0123] The technical solution of the present invention is based on the following principle. Nucleic acid of a sample to be tested is obtained. For example, target nucleic acid can be obtained by amplification method. A gRNA that can pair with the target nucleic acid is used to guide the Cas protein to recognize and bind to the target nucleic acid. Subsequently, the Cas protein activates the cleavage activity of the single-stranded nucleic acid detector, thereby cleaving the single-stranded nucleic acid detector in the system. A fluorophore and a quencher are respectively arranged at both ends of the single-stranded nucleic acid detector. If the single-stranded nucleic acid detector is cleaved, fluorescence will be excited. In other embodiments, labels capable of being detected by colloidal gold can also be arranged at both ends of the single-stranded nucleic acid detector.

[0124] Example 1: Temperature tolerance experiment of Cas12i

[0125] In this embodiment, Cas12i (shown in SEQ ID No. 3) is used, and the corresponding gRNA sequence is designed with OsTGW6 as the target nucleic acid. The single-stranded nucleic acid detector Reporter is 5’-FAM-TTGTT-3’BHQ. The reaction is carried out at different temperatures and the fluorescence signal is detected to test the temperature tolerance of Cas12i.

[0126] In the reaction system, the final concentration of Cas 12i is 100 nM, the final concentration of gRNA is 50 nM, the target nucleic acid is 500 nM, and the final concentration of Reporter (5’-FAM-TTGTT-3’BHQ) is 500 nM. The target nucleic acid is not added in the blank control.

[0127] React at 4°C - 50°C for a period of time respectively. The results are shown in Figure 1(A) - Figure 1(C). Cas12i can react and detect fluorescence signals under the conditions of 4°C - 50°C. However, at 50°C, the fluorescence signal drops sharply and the detection effect is not obvious. Among them, under the conditions of 43°C - 48°C, the detection effect is the best, and very obvious fluorescence signals can be detected within the reaction time of 2 - 5 minutes.

[0128] The inventor used Cas12a (shown in SEQ ID No.1) to conduct the above temperature tolerance experiment. The temperature it can tolerate is comparable to that of Cas12i. When the temperature exceeds 50°C, the fluorescence signal drops sharply and the detection effect is not obvious. Under the conditions of 4°C - 48°C, it can react and detect fluorescence signals.

[0129] Example 2. Temperature tolerance experiment of Cas12j

[0130] In this embodiment, Cas12j (shown in SEQ ID No.4) is used, and OsTGW6 is used as the target nucleic acid to design the corresponding gRNA sequence. The single-stranded nucleic acid detector Reporter is 5’-FAM-TTGTT-3’BHQ. React at different temperatures and detect fluorescence signals to test the temperature tolerance of Cas12j. The reaction system is the same as that in Example 1.

[0131] React at 4°C - 56°C for a period of time respectively. The results are shown in Figure 2(A) - Figure 2(C). Cas12j can react and detect fluorescence signals under the conditions of 4°C - 56°C. However, at 56°C, the fluorescence signal drops sharply and the detection effect is not obvious. Among them, under the conditions of 52°C - 54°C, the detection effect is the best, and very obvious fluorescence signals can be detected within the reaction time of 2 - 5 minutes.

[0132] Example 3. Temperature tolerance experiment of Cas12b

[0133] In this embodiment, Cas12b (shown in SEQ ID No.2) is used, and OsTGW6 is used as the target nucleic acid to design the corresponding gRNA sequence. The single-stranded nucleic acid detector Reporter is 5’-FAM-TTGTT-3’BHQ. React at different temperatures and detect fluorescence signals to test the temperature tolerance of Cas12b. The reaction system is the same as that in Example 1.

[0134] React at 4°C - 70°C for a period of time respectively. The results are shown in Figure 3(A) - Figure 3(C). Cas12b can react and detect fluorescence signals under the conditions of 4°C - 70°C. Even when the temperature exceeds 60°C (65°C, 70°C), very obvious fluorescence signals can be detected in about 2 minutes.

[0135] Example 4: Dual Detection Using Cas12i and Cas12j

[0136] In this embodiment, LAMP primers were designed for the rice OsTGW6 gene, and primers were designed for the hand, foot and mouth virus EV71 gene. Samples were loaded according to the components in Table 1, and LAMP amplification was carried out at 65°C for 30 minutes. The amplified products were run on a gel, and the electrophoresis results are as Figure 4 shown: The OsTGW6 primer set could amplify the OsTGW6 product; the EV71 primer set could amplify the EV71 product; in the same reaction system, both amplification products could be obtained simultaneously with the two primer sets.

[0137] Table 1: Components in Different LAMP Reaction Systems

[0138]

[0139] The above different LAMP amplified products were subjected to nucleic acid detection. Three experimental groups and one control group were set up, and the components of each group are shown in Table 2. The reaction temperature of each group was set to react at 48°C for 5 minutes first, and then at 52°C for 5 minutes. The reaction results are as Figure 5 shown: In the mixed sample containing OsTGW6 and EV71, the fluorescence signal is as shown in III, and the fluorescence signal continuously increases at the reaction temperatures of 48°C and 52°C; in the sample containing EV71 but not OsTGW6, the fluorescence signal is as shown in II, a weak signal is detected at 48°C, and the fluorescence signal increases sharply at 52°C; in the sample containing OsTGW6 but not EV71, the fluorescence signal is as shown in I, and the fluorescence signal is only detected at 48°C, and the fluorescence signal no longer increases when the reaction temperature is 52°C; no fluorescence signal is detected in the negative control.

[0140] Table 2: Components Added in Different Nucleic Acid Detection Systems of the Dual Detection System

[0141]

[0142]

[0143] Among them, gRNA-Cas12i is a gRNA designed for OsTGW6 based on Cas12i; gRNA-Cas12j is a gRNA designed for EV71 based on Cas12j.

[0144] The above results reflect that, by using the different temperature tolerances of Cas12i and Cas12j, dual detection can be carried out during nucleic acid detection.

[0145] Example 5: Triple detection using Cas12i, Cas12j, and Cas12b

[0146] In this embodiment, three target genes, namely ALDH, COVID, and TGW6, were amplified separately. The amplified products, Cas proteins, gRNAs, and single-stranded nucleic acid detectors were loaded according to the groups in Table 3. The reaction temperature for each group was set to react at 48 °C for 5 minutes first, then at 52 °C for 5 minutes, and finally at 60 °C for 5 minutes.

[0147] Table 3. Components added in different nucleic acid detection systems of the triple detection system

[0148]

[0149] Among them, gRNA-Cas12i is a gRNA designed based on Cas12i for ALDH; gRNA-Cas12j is a gRNA designed based on Cas12j for COVID; gRNA-Cas12b is a gRNA designed based on Cas12b for TGW6.

[0150] The reaction results are as Figure 6 shown: Compared with the sample containing only ALDH (II), the sample containing only COVID (I), and the sample containing only TGW6 (III), in the mixed sample (IV) containing ALDH + COVID + TGW6, as the reaction temperature continuously increases, the fluorescence signal will continuously increase; there are obvious differences in the fluorescence signal IV compared with I, II, and III. This reflects that, taking advantage of the differences in the temperature tolerance thresholds of Cas12i with a maximum tolerance of 50 °C, Cas12j with a maximum tolerance of 56 °C, and Cas12b that can tolerate temperatures above 60 °C, triple detection can be performed using Cas12i, Cas12j, and Cas12b.

[0151] By studying the different temperature tolerance thresholds of different Cas proteins during nucleic acid detection, the present invention can achieve multiplex nucleic acid detection of the same sample or different samples by using different combinations of Cas proteins, and has broad application prospects.

[0152] All the documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for detecting a target nucleic acid in a sample, the method being for non-disease diagnosis and treatment purposes, the method comprising contacting the sample with a nucleic acid detection composition and a single-stranded nucleic acid detector, the nucleic acid detection composition comprising a Cas protein and a gRNA; the gRNA comprising a region that binds to the Cas protein and a guide sequence that hybridizes to a target sequence on the target nucleic acid; characterized in that, The method includes subjecting the sample to any one of the following reactions (1)-(3): (1) contacting the sample with a first nucleic acid detection composition, a third nucleic acid detection composition, and a single-stranded nucleic acid detector, reacting at a first temperature for a first period of time, and then reacting at a third temperature for a third period of time; (2) contacting the sample with a first nucleic acid detection composition, a fourth nucleic acid detection composition, and a single-stranded nucleic acid detector, reacting at a first temperature for a first period of time, and then reacting at a fourth temperature for a fourth period of time; (3) contacting the sample with a first nucleic acid detection composition, a third nucleic acid detection composition, a fourth nucleic acid detection composition, and a single-stranded nucleic acid detector, reacting at a first temperature for a first period of time, then reacting at a third temperature for a third period of time, and then reacting at a fourth temperature for a fourth period of time; The first nucleic acid detection composition includes Cas12i, a first gRNA that can bind to Cas12i and hybridize with a first target sequence on the target nucleic acid; The third nucleic acid detection composition includes Cas12j, a third gRNA that can bind to Cas12j and hybridize with a third target sequence on the target nucleic acid; The fourth nucleic acid detection composition includes Cas12b, a fourth gRNA that can bind to Cas12b and hybridize with a fourth target sequence on the target nucleic acid; The first temperature is 37°C - 48°C; the third temperature is 52°C - 56°C; the fourth temperature is 60°C - 70°C; Detect the detectable signal generated by the Cas protein cleaving the single-stranded nucleic acid detector, thereby detecting the target nucleic acid.

2. The method according to claim 1, wherein The first temperature is 43°C - 48°C; the third temperature is 52°C - 54°C; the fourth temperature is 60°C - 65°C.

3. The method according to claim 1 or 2, characterized in that, The detectable signal is detected by the following methods: visual-based detection, fluorescence signal-based detection, sensor-based detection, fluorescence polarization, colloid phase transition / dispersion, electrochemical detection, and semiconductor-based detection.

4. The method according to claim 1 or 2, characterized in that, The detectable signal is detected by the following method: color detection.

5. The method according to claim 1 or 2, characterized in that, The detectable signal is detected by the following method: gold nanoparticle-based detection.

6. The method according to claim 1 or 2, characterized in that The target nucleic acid includes ribonucleotides or deoxyribonucleotides.

7. The method according to claim 6, wherein The target nucleic acid includes single-stranded nucleic acid, double-stranded nucleic acid.

8. The method according to claim 6, characterized in that The target nucleic acid includes single-stranded DNA, double-stranded DNA, single-stranded RNA.

9. The method according to claim 1 or 2, characterized in that, The target nucleic acid is viral nucleic acid, bacterial nucleic acid, specific nucleic acid different from the control.

10. The method according to claim 9, characterized in that, The specific nucleic acid different from the control is a specific mutation site.

11. The method according to claim 9, characterized in that, The virus is a plant virus or an animal virus.

12. The method according to claim 9, characterized in that The virus is papillomavirus, hepadnavirus, herpesvirus, adenovirus, poxvirus, parvovirus, coronavirus.

13. The method according to claim 12, wherein The coronavirus includes SARS, SARS-CoV2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-Cov.

14. The method according to claim 1 or 2, characterized in that, Fluorescent groups and quenching groups are respectively arranged at both ends of the single-stranded nucleic acid detector.

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