WO2022012423A1 - 利用含有无碱基间隔物的核酸检测器检测靶核酸的方法 - Google Patents

利用含有无碱基间隔物的核酸检测器检测靶核酸的方法 Download PDF

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WO2022012423A1
WO2022012423A1 PCT/CN2021/105398 CN2021105398W WO2022012423A1 WO 2022012423 A1 WO2022012423 A1 WO 2022012423A1 CN 2021105398 W CN2021105398 W CN 2021105398W WO 2022012423 A1 WO2022012423 A1 WO 2022012423A1
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nucleic acid
abasic
stranded nucleic
spacer
detector
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PCT/CN2021/105398
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French (fr)
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梁亚峰
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山东舜丰生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of nucleic acid detection, and relates to the detection of target nucleic acid using a single-stranded nucleic acid detector, in particular to a method, system and kit for detecting target nucleic acid using a nucleic acid detector containing an abasic spacer.
  • nucleic acid detection The specific detection of nucleic acid molecules (Nucleic acid detection) method has important application value, such as pathogen detection, genetic disease detection and so on.
  • pathogen detection since each pathogenic microorganism has its unique characteristic nucleic acid molecular sequence, nucleic acid molecular detection for specific species can be developed, also known as nucleic acid diagnosis (NADs, nucleic acid diagnostics). Microbial contamination detection, human pathogenic bacteria infection and other fields are of great significance.
  • 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 genome level provides a new perspective for modern molecular biology, and SNPs are closely related to biological functions, evolution, and diseases. Therefore, the development of SNPs detection and analysis technology Particularly important.
  • the currently established specific nucleic acid molecule detection usually needs to be divided into two steps, the first step is the amplification of the target nucleic acid, and the second step is the target nucleic acid detection.
  • Existing detection technologies include restriction endonuclease method, Southern, Northern, dot hybridization, fluorescent PCR detection technology, LAMP loop-mediated isothermal amplification technology, recombinase polymerase amplification technology (RPA) and other methods.
  • RPA recombinase polymerase amplification technology
  • Zhang Feng's team developed a new nucleic acid diagnostic technology (SHERLOCK technology) targeting RNA with Cas13 as the core based on RPA technology
  • the Doudna team developed a Cas12 enzyme as the core.
  • the nucleic acid probe or nucleic acid detector is the key element of the above detection technology.
  • the present invention improves the nucleic acid probe, and selects a single-stranded nucleic acid detector containing a base-free spacer as the probe. needle, thus expanding the application range of this technology.
  • the present invention provides methods, systems and kits for target nucleic acid detection using a nucleic acid detector containing an abasic spacer.
  • the present invention provides a method for detecting a target nucleic acid in a sample, the method comprising contacting the sample with a V-type CRISPR/CAS effector protein, a gRNA (guide RNA), and a single-stranded nucleic acid detector, the gRNA comprising The region bound by the CRISPR/CAS effector protein and the target sequence hybridized with the target nucleic acid; the detectable signal generated by the CRISPR/CAS effector protein cleaving the single-stranded nucleic acid detector is detected, thereby detecting the target nucleic acid; the single-stranded nucleic acid detector It is a single-stranded nucleic acid detector containing an abasic spacer.
  • the present invention also provides a reagent, system or composition for detecting target nucleic acid in a sample, the reagent, system or composition comprising V-type CRISPR/CAS effector protein, gRNA (guide RNA) and a single A stranded nucleic acid detector, the gRNA includes a region that binds to the CRISPR/CAS effector protein and a guide sequence that hybridizes with a target nucleic acid; the single-stranded nucleic acid detector is a single-stranded nucleic acid detector containing an abasic spacer.
  • the present invention also provides a kit for detecting target nucleic acid in a sample, the kit comprising a V-type CRISPR/CAS effector protein, a gRNA (guide RNA) and a single-stranded nucleic acid detector, the gRNA It includes a region that binds to the CRISPR/CAS effector protein and a guide sequence that hybridizes to the target nucleic acid; the single-stranded nucleic acid detector is a single-stranded nucleic acid detector containing an abasic spacer.
  • the present invention also provides the application of the above reagent, system, composition or kit in detecting target nucleic acid in a sample.
  • the present invention also provides the application of V-type CRISPR/CAS effector protein in detecting target nucleic acid in a sample.
  • the present invention also provides the application of the above-mentioned CRISPR/CAS effector protein, gRNA (guide RNA) and single-stranded nucleic acid detector in detecting target nucleic acid in a sample, or preparing a reagent for detecting target nucleic acid in a sample , compositions, systems or kits.
  • the V-type CRISPR/CAS effector protein after the V-type CRISPR/CAS effector protein is combined with or hybridized with the target nucleic acid in the sample, it can cut the single-stranded nucleic acid detector in the system; the single-stranded nucleic acid detector contains abasic nucleic acid. Spacer-based single-stranded nucleic acid detector.
  • the present invention also provides the application of the V-type CRISPR/CAS effector protein in preparing a reagent for detecting target nucleic acid in a sample.
  • the single-stranded nucleic acid detector is a single-stranded nucleic acid detector containing an abasic spacer, and the single-stranded nucleic acid detector contains at least one arbitrary nucleotide (arbitrary nucleotide is abbreviated as N).
  • the at least 1 arbitrary nucleotide includes 1-25 arbitrary nucleotides, for example, 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 arbitrary nucleotides) and at least 1 arbitrary abasic spacer (arbitrary abasic spacer is abbreviated as S, specifically can be one or any of dSpacer(abasic furan, dS), Spacer C3, Spacer C6, Spacer C12, Spacer9, Spacer12, Spacer18, Inverted Abasic Site(dSpacer abasic furan) and rAbasic Site(rSpacer abasic furan) , the at least 1 arbitrary abasic spacer includes 1-25 abasic spacers, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 None base spacers
  • the single-stranded nucleic acid detector contains at least one arbitrary nucleotide and at least one arbitrary abasic spacer.
  • the single-stranded nucleic acid detector contains one arbitrary nucleotide, and at least one end of the one arbitrary nucleotide is connected to at least one arbitrary abasic spacer, such as a single-stranded nucleic acid
  • the detector sequence is NS, NSS, NSSS, SN, SSN or SSSN;
  • the two ends of the one arbitrary nucleotide are respectively connected with at least one arbitrary abasic spacer, for example, the single-stranded nucleic acid detector sequence is SNS, SSNSS, SNSS or SSNS;
  • the two ends of the one arbitrary nucleotide are respectively connected with two arbitrary abasic spacers, and the sequence of the single-stranded nucleic acid detector is SSNSS.
  • the single-stranded nucleic acid detector has at least 2 consecutive nucleotides, and the at least 2 consecutive nucleotides include 2-25 consecutive nucleotides, eg, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 1, 21, 22, 23, 24, 25 consecutive nucleotides with at least 1 abasic spacer at one end of the at least 2 consecutive nucleotides, eg, 1-25 Abasic spacers, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 abasic spacers; if the sequence is NNS, SNN, NNSS or SSNN, preferably The single-stranded nucleic acid detector sequence is C3CT, CTC3, C6CT, CTC6, TGSS, SSTS, CTS or SCT.
  • the sequence is SNNS, SSNNS, SNNSS or SSNNSS
  • the single-stranded nucleic acid detector sequence includes SCTS , SSTGS, STGSS, SSAASS, SSACSS, SSAGSS, SSATSS, SSTASS, SSTCSS, SSTGSS, SSTTSS, SSCASS, SSCCSS, SSCGSS, SSCTSS, SSGASS, SSGCSS, SSGGSS, and SSGTSS.
  • the single-stranded nucleic acid detector contains at least 2 discontinuous nucleotides with at least one abasic spacer between the at least 2 discontinuous nucleotides, eg, the sequence includes at least NSN One or more of , NSSN or NSSSN, preferably, such as sequences such as NSN, NSSN or NSSSN, preferably, the single-stranded nucleic acid detector sequence is ASST, TSSA, ASSA, TSSST, TSST or TST.
  • the at least 1 abasic spacer includes 1-25 abasic spacers, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 abases spacer.
  • the single-stranded nucleic acid detector is a single-stranded nucleic acid detector containing an abasic spacer, and the single-stranded nucleic acid detector contains at least one arbitrary nucleotide and at least one nonbasic spacer.
  • Base spacer preferably, the single-stranded nucleic acid detector does not include any two consecutive nucleotides.
  • does not include any two consecutive nucleotides can be understood as, two adjacent nucleotides are connected by at least 1 abasic spacer, for example, 1-20 abasic spacers; another example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 abasic spacers.
  • both ends of any single nucleotide in the single-stranded nucleic acid detector are respectively connected with at least one abasic spacer; more preferably, the two ends of any single nucleotide are respectively connected with At least 2 abasic spacers.
  • the single-stranded nucleic acid detector contains only one arbitrary nucleotide and at least one abasic spacer; preferably, the two ends of the nucleotide are respectively connected with at least one Abasic spacer; more preferably, at least two abasic spacers are connected to both ends of the nucleotide.
  • the two ends of the nucleotide are respectively connected with 2-20 abasic spacers; preferably, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 abasic spacers.
  • the single-stranded nucleic acid detector has 1-300 random nucleotides and 1-300 abasic spacers, preferably, 1-200 nucleotides and 1-200 abasic spacers Base spacer, preferably, 1-100 nucleotides and 1-100 abasic spacers, preferably, with 1-30 nucleotides and 1-30 abasic spacers, more preferably, 1 Nucleotides and 2-15 abasic spacers.
  • the single-stranded nucleic acid detector contains two consecutive arbitrary nucleotides and at least two abasic spacers, and both ends of the two consecutive arbitrary nucleotides are respectively connected with At least 1 abasic spacer. Further, in the single-stranded nucleic acid detector, at least two abasic spacers are respectively connected to both ends of the two consecutive arbitrary nucleotides. In one embodiment, 2-20 abasic spacers are respectively connected to both ends of the two consecutive arbitrary nucleotides; preferably, 2, 3, 4, 5, 6, 7 1, 8, 9, 10, 15 or 20 abasic spacers.
  • the single-stranded nucleic acid detector further contains at least one additional group of nucleotides, and two ends of each additional group of nucleotides are respectively connected with at least one abasic spacer, so that The additional nucleotides are 1 arbitrary nucleotide or 2 consecutive arbitrary nucleotides.
  • the single-stranded nucleic acid detector is composed of two consecutive random nucleotides and an abasic spacer, and two ends of the two consecutive random nucleotides are respectively connected with two free nucleotides. base spacers.
  • nucleotides in the single-stranded nucleic acid detector are ribonucleotides and/or deoxyribonucleotides; the bases of the ribonucleotides are selected from one of A, U, C, and G. one or any of several kinds; the base of the deoxyribonucleotide is selected from one or any of several kinds of A, T, C, and G.
  • the two consecutive arbitrary nucleotides are selected from the group consisting of AA, AT, AC, AG, TT, TA, TC, TG, CC, CA, CT, CG, GG, GA, GT, GC , UU, UA, UC, UG, AU, CU or GU; wherein, A, C, G can be ribonucleotides or deoxyribonucleotides.
  • the single-stranded nucleic acid detector has 2-300 arbitrary nucleotides and 2-300 abasic spacers, preferably, 2-200 nucleotides and 2-200 abasic spacers Base spacers, preferably, 2-100 nucleotides and 2-100 abasic spacers, preferably, 2-30 nucleotides and 1-30 abasic spacers, more preferably, 2 Nucleotides and 2-15 abasic spacers.
  • the abasic spacer is selected from dSpacer (abasic furan), Spacer C3 (C3), Spacer C6 (C6), Spacer C12, Spacer9, Spacer12, Spacer18, Inverted Abasic Site (dSpacer abasic furan) and rAbasic One or any of the Site (rSpacer abasic furan).
  • the abasic spacer is one or more of dSpacer, Spacer C3(C3) or Spacer C6(C6).
  • the abasic spacer is a dSpacer.
  • dSpacer is not only structurally very similar to the natural site, but also quite stable. The structure looks like this:
  • the Spacer C3 (C3) structure is shown in the following figure:
  • the Spacer C6 (C6) structure is shown in the following figure:
  • nucleotides are ribonucleotides and/or deoxyribonucleotides; the bases of the ribonucleotides are selected from one or any of A, U, C, G, T, and I several; the base of the deoxyribonucleotide is selected from one or any of several A, T, C, G, U, and I.
  • the single-stranded nucleic acid detector does not hybridize with the gRNA.
  • the V-type CRISPR/CAS effector protein is selected from Cas12, Cas14 family proteins or mutants thereof; in one embodiment, the Cas protein is preferably the Cas12 family, including but not limited to Cas12a, Cas12b, Cas12i, Cas12j One or any of several; the Cas14 family proteins are selected from Cas14a and/or Cas14b.
  • the Cas12a is selected from one or any of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a;
  • the Cas12a is preferably LbCas12a, and the amino acid sequence is as shown in SEQ ID As shown in No.1, or, the amino acid sequence shown in SEQ ID No.1 or its active fragment is passed through one or more (such as 2, 3, 4, 5, 6, 7, 8, A derivative protein formed by substitution, deletion or addition of 9 or 10) amino acid residues and having substantially the same function.
  • the amino acid sequence of Cas12b is shown in SEQ ID No. 2, or, the amino acid sequence shown in SEQ ID No. 2 or its active fragment is subjected to one or more (such as 2, 3 , 4, 5, 6, 7, 8, 9 or 10) amino acid residues are formed by substitution, deletion or addition of amino acid residues and have substantially the same function.
  • the amino acid sequence of Cas12i is shown in SEQ ID No. 3, or, the amino acid sequence shown in SEQ ID No. 3 or its active fragment is passed through one or more (such as 2, 3 , 4, 5, 6, 7, 8, 9 or 10) amino acid residues are formed by substitution, deletion or addition of amino acid residues and have substantially the same function.
  • the amino acid sequence of Cas12j is shown in SEQ ID No. 4, or, the amino acid sequence shown in SEQ ID No. 4 or its active fragment is passed through one or more (such as 2, 3 , 4, 5, 6, 7, 8, 9 or 10) amino acid residues are formed by substitution, deletion or addition of amino acid residues and have substantially the same function.
  • the Cas protein mutant comprises amino acid substitutions, deletions or substitutions, and the mutant retains at least its trans cleavage activity.
  • the mutant has Cis and trans cleavage activities.
  • the detectable signal is achieved by the following methods: vision-based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization, fluorescence signal detection, colloidal phase transition/dispersion, electrochemical Inspection and semiconductor-based inspection.
  • the methods of the invention further comprise the step of measuring the detectable signal produced by the CRISPR/CAS effector protein (Cas protein).
  • the Cas protein can stimulate the cleavage activity of the single-stranded nucleic acid after recognizing the target nucleic acid or hybridizing with the target nucleic acid, thereby cleaving the single-stranded nucleic acid detector to generate a detectable signal.
  • the detectable signal may be any signal generated when the single-stranded nucleic acid detector is cleaved.
  • the detectable signal may be read out by any suitable means, including but not limited to: measurement of a detectable fluorescent signal, gel electrophoresis detection (by detecting changes in bands on the gel), visual or sensor-based color Detection of presence or absence, or differences in color presence (eg, based on gold nanoparticles) and differences in electrical signal.
  • the detectable signal is achieved by the following manner: the 5' end and the 3' end of the single-stranded nucleic acid detector are respectively provided with different reporter groups, when the single-stranded nucleic acid detector is cleaved After cleavage, a detectable reporter signal can be displayed; for example, a fluorescent group and a quenching group are set at both ends of the single-stranded nucleic acid detector, and when the single-stranded nucleic acid detector is cleaved, a detectable report signal can be displayed. fluorescence signal.
  • the fluorescent group is selected from one or any of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460; the quenching group is selected from One or any of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.
  • the detectable signal can also be achieved by the following methods: the 5' end and the 3' end of the single-stranded nucleic acid detector are respectively provided with different label molecules, and the reaction signal is detected by colloidal gold detection. .
  • the target nucleic acid includes ribonucleotides or deoxyribonucleotides, including single-stranded nucleic acid and double-stranded nucleic acid, such as single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA.
  • the target nucleic acid includes DNA, RNA, preferably single-stranded nucleic acid or double-stranded nucleic acid or nucleic acid modification.
  • the target nucleic acid is derived from samples such as viruses, bacteria, microorganisms, soil, water sources, human body, animals, plants and the like.
  • 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 and the like.
  • the method further comprises the step of obtaining the target nucleic acid from the sample.
  • the target nucleic acid is viral nucleic acid, bacterial nucleic acid, disease-related specific nucleic acid, such as a specific mutation site or SNP site or a nucleic acid that is different from a control;
  • the virus is a plant Virus or animal virus, for example, papilloma virus, hepadnavirus, herpes virus, adenovirus, poxvirus, parvovirus, coronavirus;
  • the virus is a coronavirus, preferably SARS, SARS-CoV2 (COVID-19) -19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-Cov.
  • the target nucleic acid is derived from a cell, eg, from a cell lysate.
  • the measurement of the detectable signal may be quantitative, and in other embodiments, the measurement of the detectable signal may be qualitative.
  • the single-stranded nucleic acid detector generates a first detectable signal before being cleaved by the Cas protein, and generates a second detectable signal different from the first detectable signal after being cleaved.
  • the gRNA includes a sequence targeting the target nucleic acid (target sequence) and a sequence recognizing Cas protein (direct repeat sequence or part thereof).
  • the guide sequence includes 10-40bp; preferably, 12-25bp; preferably, 15-23bp; preferably, 16-18bp.
  • the molar ratio of the amount of Cas protein to gRNA is (0.8-1.2):1.
  • the final concentration of the Cas protein is 20-200nM, preferably, 30-100nM, more preferably, 40-80nM, more preferably, 50nM.
  • the gRNA is used in a final concentration of 20-200 nM, preferably, 30-100 nM, more preferably, 40-80 nM, more preferably, 50 nM.
  • the target nucleic acid is used at a final concentration of 5-100 nM, preferably, 10-50 nM.
  • the single-stranded nucleic acid detector is used at a final concentration of 100-1000nM, preferably, 150-800nM, preferably, 200-800nM, preferably, 200-500nM, preferably, 200-300nM.
  • hybrid or “complementary” or “substantially complementary” mean that a nucleic acid (eg, RNA, DNA) comprises a nucleotide sequence that enables it to bind non-covalently, i.e. in a sequence-specific, anti-parallel fashion ( That is, a nucleic acid that specifically binds a complementary nucleic acid) forms base-pairing and/or G/U base-pairing, "anneals” or “hybridizes” with another nucleic acid. Hybridization requires that the two nucleic acids contain complementary sequences, although there may be mismatches between the bases.
  • hybridizable nucleic acids are 8 nucleotides or more in length (eg, 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more) nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).
  • sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to hybridize specifically.
  • Polynucleotides may comprise 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 the sequence complementarity of the target region in the target nucleic acid sequence to which it hybridizes is 100%.
  • amino acid refers to a carboxylic acid containing an amino group.
  • Various proteins in living organisms are composed of 20 basic amino acids.
  • nucleic acid refers to DNA, RNA, or hybrids thereof, which may be double-stranded or single-stranded.
  • homology or “identity” are used to refer to the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by an adenine, or both A position in each of the polypeptides is occupied by a lysine)
  • the molecules are identical at that position. between the two sequences.
  • comparisons are made when two sequences are aligned for maximum identity. Such alignment can be accomplished by using, for example, amino acid sequence identity by conventional methods, see for example Smith and Waterman, 1981, Adv. Appl. Math.
  • CRISPR refers to clustered regularly interspaced short palindromic repeats (Clustered regularly interspaced short palindromic repeats) derived from the immune system of microorganisms.
  • biotin also known as vitamin H
  • vitamin H is a small molecule vitamin with a molecular weight of 244 Da.
  • vidin also known as avidin
  • avidin is a basic glycoprotein with 4 binding sites with extremely high affinity for biotin. Commonly used avidin is streptavidin. The extremely strong affinity of biotin to avidin can be used to amplify or enhance the detection signal in detection systems.
  • biotin is easily combined with proteins (such as antibodies, etc.) by covalent bonds, and the avidin molecules combined with enzymes react with biotin molecules combined with specific antibodies, which not only plays a multi-stage amplification role, but also because of When the enzyme encounters the catalysis of the corresponding substrate, it becomes colored, and the purpose of detecting the unknown antigen (or antibody) molecule is achieved.
  • the single-stranded nucleic acid detector of the present invention is used in the detection method or system to report whether the target sequence is contained.
  • the two ends of the single-stranded nucleic acid detector include different reporter groups or label molecules. When it is in an initial state (ie, when it is not in a cleaved state), it does not present a report signal. When the single-stranded nucleic acid detector is cleaved, it presents A detectable signal is detected, that is, a detectable difference is exhibited after cleavage and before cleavage.
  • a detectable difference if a detectable difference can be detected, it reflects that the target nucleic acid contains the target sequence to be detected; or, if the detectable difference cannot be detected, it reflects that the target nucleic acid does not contain the target sequence to be detected. target sequence.
  • the reporter group or labeling molecule includes a fluorescent group and a quenching group
  • the fluorescent group is selected from FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red Or one or any of LC RED460
  • the quenching group is selected from one or any of BHQ1, BHQ2, BHQ3, Dabcyl or Tamra.
  • the single-stranded nucleic acid detector has a first molecule (such as FAM or FITC) attached to the 5' end and a second molecule (such as biotin) attached to the 3' end.
  • the reaction system containing the single-stranded nucleic acid detector is used in conjunction with a flow strip to detect the characteristic sequence (preferably, colloidal gold detection method).
  • the flow strip is designed to have two capture lines, an antibody that binds to the first molecule (ie, the first molecule antibody) is provided at the sample contact end (colloidal gold), and the first line (control line) contains the first molecule that binds to the first molecule.
  • an antibody of a molecule of antibody that contains a second molecule of antibody bound to a second molecule at the test line ie, a second molecule of antibody, such as avidin.
  • the first molecule of antibody binds to the first molecule carrying cleaved or uncleaved oligonucleotides to the capture line, where the cleaved reporter will bind to the first molecule of antibody at the first capture line antibody, while the uncleaved reporter will bind the second molecule of antibody at the second capture line. Binding of the reporter group at each line will result in a strong readout/signal (eg color). As more reporters are cleaved, more signal will accumulate at the first capture line, and less signal will appear at the second line.
  • the present invention relates to the use of a flow strip as described herein for the detection of nucleic acids.
  • the invention relates to a method for the detection of nucleic acids using a flow strip as defined herein, such as a (lateral) flow assay or a (lateral) flow immunochromatographic assay.
  • the molecules in the single-stranded nucleic acid detector can be replaced with each other, or the positions of the molecules can be changed, as long as the reporting principle is the same as or similar to the present invention, the improved methods are also included in the present invention.
  • the detection method of the present invention can be used for quantitative detection of characteristic sequences to be detected.
  • the quantitative detection index can be quantified according to the signal strength of the reporter group, for example, according to the luminescence intensity of the fluorophore, or according to the width of the color band.
  • an "abasic spacer (Spacer, abbreviated as S in the present invention) refers to a nucleoside that does not contain specific coding information. Abasic spacers can be attached to oligonucleotides, including at the 3' and 5' ends, or within the nucleotide chain. Common Spacers include: dSpacer(abasic furan), Spacer C3, Spacer C6, Spacer C12, Spacer 9, Spacer12, Spacer18, Inverted Abasic Site(dSpacer abasic furan) and rAbasic Site(rSpacer abasic furan).
  • abasic spacer is abasic spacer known in the art, for example, U.S. Patent US8153772B2 discloses dSpacer, Spacer 9, Spacer 18, Spacer C3; Chinese patent application CN101454451A discloses dSpacer.
  • dSpacer The preferred abasic spacer "dSpacer” herein is also referred to as an abasic site, abbreviated in the present invention as dS, tetrahydrofuran (THF) or apurine/apyrimidinic (AP) site), or, an abasic linker in which the methylene group is located at position 1 of the 2'-deoxyribose.
  • dSpacer is not only structurally very similar to the natural site, but also quite stable. The structure looks like this:
  • dSpacer When the dSpacer is connected to nucleotides, it can form the following structure:
  • Cas protein refers to a CRISPR-associated protein, preferably from a type V or type VI CRISPR/CAS protein, which once binds to the characteristic sequence (target sequence) to be detected (i.e., forms a Cas protein-gRNA-target sequence triplet) metacomplex), can induce its trans activity, ie, random cleavage of non-targeting single-stranded nucleotides (ie, the single-stranded nucleic acid detectors described herein).
  • Cas protein When Cas protein binds to the characteristic sequence, it can induce its trans activity by cutting or not cutting the characteristic sequence; preferably, it induces its trans activity by cutting the characteristic sequence; more preferably, it induces its trans activity by cutting the single-chain characteristic sequence. trans activity.
  • the Cas protein recognizes the characteristic sequence by recognizing the PAM (protospacer adjacent motif) adjacent to the characteristic sequence.
  • the Cas protein of the present invention is a protein with at least trans cleavage activity, preferably, the Cas protein is a protein with Cis and trans cleavage activities.
  • the Cis activity refers to the activity of Cas protein that can recognize the PAM site and specifically cleave the target sequence under the action of gRNA.
  • the Cas proteins described in the present invention include V-type CRISPR/CAS effector proteins, including Cas12, Cas14 and other protein families.
  • Cas12 protein such as Cas12a, Cas12b, Cas12i, Cas12j
  • the Cas protein is Cas12a, Cas12b, Cas12i, Cas12j
  • Cas14 protein family includes Cas14a, Cas14b and the like.
  • Cas proteins referred to herein, such as Cas12 also encompass functional variants of Cas or homologs or orthologs thereof.
  • a "functional variant" of a protein as used herein refers to a variant of such a protein that at least partially retains the activity of the protein. Functional variants may include mutants (which may be insertion, deletion or substitution mutants), including polymorphs and the like. Also included in functional variants are the fusion products of such a protein with another normally unrelated nucleic acid, protein, polypeptide or peptide. Functional variants may be naturally occurring or may be man-made. Advantageous embodiments may involve engineered or non-naturally occurring V-type DNA targeting effector proteins.
  • one or more nucleic acid molecules encoding a Cas protein can be codon-optimized for expression in eukaryotic cells.
  • Eukaryotes can be as described herein.
  • One or more nucleic acid molecules may be engineered or non-naturally occurring.
  • the Cas12 protein, or an ortholog or homolog thereof may contain one or more mutations (and thus the nucleic acid molecule encoding it may have one or more mutations.
  • the mutations may be artificially introduced mutations and may Including, but not limited to, one or more mutations in the catalytic domain.
  • the Cas protein can be from: Ciliary, Listeria, Corynebacterium, Saterella, Legionella, Treponema, Progenitor, Eubacteria, Streptococcus , Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Azospirillum, Sphaerochaeta, Gluconacetobacter, Neisseria, Roche, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma , Campylobacter and Lachnospira.
  • the Cas protein is selected from the proteins consisting of the following sequences:
  • the Cas protein further comprises 50%, preferably 55%, preferably 60%, preferably 65%, preferably 70%, preferably 75%, preferably 80%, preferably 85%, preferably 90% of the above sequence , preferably 95%, sequence identity, and transactive proteins.
  • the Cas protein can be obtained by recombinant expression vector technology, that is, the nucleic acid molecule encoding the protein is constructed into a suitable vector, and then transformed into a host cell, so that the encoding nucleic acid molecule is expressed in the cell, thereby obtaining the corresponding protein.
  • the protein can be secreted by cells, or the protein can be obtained by breaking cells through conventional extraction techniques.
  • the encoding nucleic acid molecule may be integrated into the genome of the host cell for expression, or may not be integrated into the host cell for expression.
  • the vector further includes regulatory elements that facilitate sequence integration or self-replication.
  • the vector can be, for example, plasmid, virus, cosmid, phage, etc., which are well known to those skilled in the art.
  • the expression vector in the present invention is a plasmid.
  • the vector further comprises one or more regulatory elements selected from the group consisting of promoters, enhancers, ribosome binding sites for translation initiation, terminators, polyadenylation sequences, and selectable marker genes.
  • Host cells can be prokaryotic cells, such as E. coli, Streptomyces, Agrobacterium; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. It will be clear to those of ordinary skill in the art how to select appropriate vectors and host cells.
  • gRNA is also referred to as guide RNA or guide RNA, and has the meaning commonly understood by those skilled in the art.
  • guide RNAs may comprise direct repeats and guide sequences, or consist essentially of or consist of direct repeats and guide sequences (also referred to as spacers in the context of endogenous CRISPR systems) (spacer)) composition.
  • gRNAs can include crRNA and tracrRNA, or only crRNA, depending on the Cas protein they depend on.
  • crRNA and tracrRNA can be artificially fused to form single guide RNA (sgRNA).
  • a targeting sequence is any sequence that is sufficiently complementary to a target sequence (characterized in the present invention) to hybridize to the target sequence and direct specific binding of the CRISPR/Cas complex to the target sequence.
  • a polynucleotide sequence typically having a sequence length of 12-25 nt.
  • the direct repeats can be folded to form specific structures (eg, stem-loop structures) for Cas protein recognition to form complexes.
  • the targeting sequence need not be 100% complementary to the characteristic sequence (target sequence).
  • the targeting sequence is not complementary to the single-stranded nucleic acid detector.
  • the degree of complementarity (match) between a target 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 ability of one of ordinary skill in the art. For example, there are published and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython, and SeqMan.
  • the gRNA of the present invention can be natural, or it can be artificially modified or designed and synthesized.
  • 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 unicellular organisms such as bacteria, yeast, protozoa and amoeba, etc., multicellular organisms such as plants or animals, Included are samples from healthy or apparently healthy human subjects or human patients affected by the condition or disease to be diagnosed or investigated, such as infection by pathogenic microorganisms such as pathogenic bacteria or viruses).
  • a biological sample can be obtained from, for example, blood, plasma, serum, urine, feces, sputum, mucus, lymph, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous fluid, or any biological fluid obtained from bodily secretions, exudates, exudates (for example, fluids obtained from an abscess or any other site of infection or inflammation) or from joints (for example, normal joints or joints affected by disease, such as rheumatoid arthritis, osteoarthritis, gout, or septic arthritis), or swabs from skin or mucosal surfaces.
  • a sample may also be a sample obtained from any organ or tissue (including biopsy or autopsy specimens, such as tumor biopsies) or may contain cells (primary or cultured) or cultures conditioned by any cell, tissue or organ base.
  • Exemplary samples include, but are not limited to, cells, cell lysates, blood smears, cytocentrifugation preparations, cytology smears, body fluids (eg, blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc. ), tissue biopsies (eg, tumor biopsies), fine needle aspirates, and/or tissue sections (eg, cryostat tissue sections and/or paraffin-embedded tissue sections).
  • the biological sample can be a plant cell, callus, tissue or organ (eg, root, stem, leaf, flower, seed, fruit), and the like.
  • the target nucleic acid also includes DNA molecules formed by reverse transcription of RNA.
  • the target nucleic acid can be amplified by techniques known in the art, and the amplification technique is isothermal amplification.
  • isothermal amplification can be nucleic acid sequencing-based amplification (NASBA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA) ), helicase-dependent amplification (HDA), or nickase amplification reaction (NEAR).
  • NASBA nucleic acid sequencing-based amplification
  • RPA recombinase polymerase amplification
  • LAMP loop-mediated isothermal amplification
  • SDA strand displacement amplification
  • HDA helicase-dependent amplification
  • NEAR nickase amplification reaction
  • non-isothermal amplification methods may be used, including but not limited to PCR, multiple displacement amplification (MDA), rolling circle amplification (RCA), ligase chain reaction (LCR), or derivatization Biological Amplification Method (RAM).
  • MDA multiple displacement amplification
  • RCA rolling circle amplification
  • LCR ligase chain reaction
  • RAM derivatization Biological Amplification Method
  • the detection method of the present invention further includes the step of amplifying the target nucleic acid; the detection system further includes a reagent for amplifying the target nucleic acid.
  • the amplified reagents include one or more of the following group: DNA polymerases, strand displacement enzymes, helicases, recombinases, single-stranded binding proteins, and the like.
  • the target nucleic acid can be obtained by the method of amplification
  • the Cas protein can be used to guide the Cas protein to recognize and bind to the target nucleic acid by pairing with the target nucleic acid;
  • Excite the cutting activity of the single-stranded nucleic acid detector thereby cutting the single-stranded nucleic acid detector in the system;
  • the two ends of the single-stranded nucleic acid detector are respectively provided with a fluorescent group and a quenching group, if the single-stranded nucleic acid detector is cut, then Fluorescence will be excited; if the single-stranded nucleic acid detector cannot be cleaved, the fluorescence will not be excited; in other embodiments, the two ends of the single-stranded nucleic acid detector can also be provided with labels that can be detected by colloidal gold.
  • Example 1 Detection of target nucleic acid using a nucleic acid detector containing an abasic spacer (SSNSS)
  • a nucleic acid detector containing an abasic spacer having a structure of dSpacer-dSpacer-N-dSpacer-dSpacer is used as a detection probe, wherein N is any deoxyribonucleotide, and dSpacer is abbreviated as dS.
  • the nucleic acid detector probe sequence containing an abasic spacer is 5'6-FAM/dS//dS//A///dS//dS//3'BHQ1, 5'6-FAM/dS// dS//T//dS//dS//3'BHQ1, 5'6-FAM/dS//dS//C//dS//dS//3'BHQ1 or 5'6-FAM/dS/// dS//G//dS//dS//3'BHQ1; the abasic spacer is selected as dSpacer, and the A, T, C, and G are DNA.
  • the applicant has verified the detection effect of Cas12a (SEQ ID No. 1), Cas12b (SEQ ID No. 2) and Cas12j (SEQ ID No. 4) when using the nucleic acid detector containing the above-mentioned abasic spacer , the experimental design is as follows:
  • LbCas12a-TGW6-g1 The sequence of above-mentioned LbCas12a-TGW6-g1 is shown in SEQ ID No.7;
  • AaCas12b-TGW6-g1 The sequence of above-mentioned AaCas12b-TGW6-g1 is shown in SEQ ID No.8;
  • the content of each component in the 20 microliter system is as follows:
  • Example 2 When Cas12i is selected as the Cas effector protein, the detection of target nucleic acid is carried out using a nucleic acid detector containing an abasic spacer (SSNSS)
  • Select sequence is 5'6-FAM/dS//dS//N//dS//dS//3'BHQ1 single-stranded nucleic acid detector, described abasic spacer is selected as dSpacer, according to embodiment 1
  • the experimental system verified Cas12i shown in SEQ ID No.3, and in some cases, also referred to by Cas12i3
  • the target nucleic acid was Cas12i3-g2-ssDNA0
  • the gRNA was DRi3-gOsTGW6-2 (sequence is agagaaugugugcauagucacacuuucaccgacagcagcaugaacu).
  • the activated bypass activation activity can cut the sequence to 5'6-FAM/dS//dS//N//dS//dS//3'BHQ1 (without The base spacer is selected as a single-stranded nucleic acid detector of dSpacer, that is, Cas12i can also cut a single-stranded nucleic acid detector containing a base spacer.
  • Embodiment 3 Contrast the influence of two consecutive arbitrary nucleotides and two consecutive arbitrary nucleotides connected with base spacers at both ends on the detection effect (NN vs. SSNNSS)
  • a nucleic acid detector with a sequence of two consecutive arbitrary nucleotides is used to detect the target nucleic acid, and the specific two consecutive arbitrary nucleotides are TT, TG, CA and CT respectively; A quencher group and a fluorophore are connected to the ends, respectively.
  • a nucleic acid detector containing an abasic spacer with a structure of 5'6-FAM//S//S//N//N//S//S//3'BHQ1 is also used as a detection method.
  • Probe wherein the abasic spacer is selected as dSpacer (dS), N//N is 2 consecutive arbitrary nucleotides, and the specific sequence of N//N is AA, AT, AC, AG, TA, TT , TC, TC, CA, CT, CC, CG, GA, GT, GC and GG, the A, T, C, G are DNA.
  • the applicant takes a total of 20 single-stranded nucleic acid detectors with the above-mentioned sequences as NN and SSNNSS, for Cas12a (SEQ ID No. 1), Cas12b (SEQ ID No. 2) and Cas12i (SEQ ID No. 3), according to the examples
  • the target nucleic acid and gRNA corresponding to the Cas protein in 1 were verified with a 20 ⁇ l system.
  • Cas12a SEQ ID No. 1
  • Cas12b SEQ ID No. 2
  • Cas12i SEQ ID No. 3
  • Example 4 Comparison of the influence of the difference in the number of abasic spacers connected at both ends of two consecutive arbitrary nucleotides on the detection effect (SNNS, SNNSS, SSNNS)
  • the number of abasic spacers connected at both ends was adjusted.
  • synthetic The single-stranded nucleic acid detector with the sequence of dS//C//T//dS, dS//dS//T//G//dS (the base-free spacer is selected as dSpacer, and the two ends are connected with quenchers respectively group and fluorophore), the detection was still carried out according to the system in the previous example.
  • Example 5 The influence of two consecutive arbitrary nucleotides with only one end (3' end or 5' end) connected to an abasic spacer on the detection effect (NNS, NNSS, SNN, SSNN)
  • single-stranded nucleic acid detectors with different sequences of nucleotides and different combinations of abasic spacers were synthesized for verification.
  • the bypass cleavage activity of the effector protein cleaves, showing a fluorescent signal.
  • Example 6 Influence on the detection effect by connecting different numbers of abasic spacers between discontinuous arbitrary nucleotides
  • the single-stranded nucleic acid detector contains two or more arbitrary nucleotides, and a base-free spacer is included between the nucleotides, it can also be used as a single-stranded nucleic acid detector; No effect on the number of base spacers.

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Abstract

本发明提供了一种利用含有无碱基间隔物的单链核酸检测器进行靶核酸检测的方法、系统和试剂盒，所述的检测方法包括向含有靶核酸的反应体系中加入gRNA、Cas蛋白和单链核酸检测器，所述单链核酸检测器含有至少1个任意的核苷酸和至少1个无碱基间隔物。

Description

利用含有无碱基间隔物的核酸检测器检测靶核酸的方法 技术领域
本发明涉及核酸检测领域，涉及利用单链核酸检测器进行靶核酸的检测，尤其涉及一种利用含有无碱基间隔物的核酸检测器检测靶核酸的方法、系统和试剂盒。
背景技术
特异性检测核酸分子(Nucleic acid detection)方法具有重要的应用价值，例如病原体的检测，遗传病检测等。在病原体检测方面，由于每种病原体微生物都有其独一无二的特征核酸分子序列，因此可以开发出针对特定物种的核酸分子检测，也称为核酸诊断(NADs,nucleic acid diagnostics)，在食品安全、环境微生物污染检测，人体病原菌感染等领域具有重要义。另一个方面是对人或其他物种的单核苷酸多态性(SNPs,single nucleotide polymorphisms)的检测。在基因组水平上去理解遗传变异和生物学功能之间的关系为现代分子生物学提供了新视角，而其中SNPs对生物学的功能、进化和疾病等密切相关，因此SNPs的检测与分析技术的发展尤为重要。
目前建立的特异性核酸分子检测通常需要分为两步，第一步是目的核酸的扩增，第二步是目的核酸检测。现有检测技术包括限制性核酸内切酶方法、Southern、Northern、斑点杂交、荧光PCR检测技术、LAMP环介导等温扩增技术、重组酶聚合酶扩增技术(RPA)等方法。2012年之后，CRISPR基因编辑技术兴起，张锋团队基于RPA技术开发了一种以Cas13为核心的靶向RNA的新核酸诊断技术(SHERLOCK技术)，Doudna团队开发了一种以Cas12酶为核心的诊断技术(DETECTR技术),中国科学院上海植物生理生态研究所王金博士等也开发了一种基于Cas12的新型核酸检测技术(HOLMES技术)。基于CRISPR技术开发的核酸检测技术正在日益发挥重要作用。
在基于CRISPR的核酸检测技术中，核酸探针或者核酸检测器是上述检测技术的关键元件，本发明对核酸探针进行了改进，选择了含有无碱基间隔物的单链核酸检测器作为探针，从而扩展了该技术的应用范围。
发明内容
本发明提供了利用含有无碱基间隔物的核酸检测器进行靶核酸检测的方法、系统和试剂盒。
方法
一方面，本发明提供了一种检测样品中靶核酸的方法，所述方法包括将样品与V型CRISPR/CAS效应蛋白、gRNA(指导RNA)和单链核酸检测器接触，所述gRNA包括与所述CRISPR/CAS效应蛋白结合的区域和与靶核酸杂交的导向序列；检测由CRISPR/CAS效应蛋白切割单链核酸检测器产生的可检测信号，从而检测靶核酸；所述单链核酸检测器为含有无碱基间隔物的单链核酸检测器。
系统或组合物、试剂盒
另一方面，本发明还提供了一种用于检测样品中靶核酸的试剂、系统或组合物，所述试剂、系统或组合物包括V型CRISPR/CAS效应蛋白、gRNA(指导RNA)和单链核酸检测器，所述gRNA包括与所述CRISPR/CAS效应蛋白结合的区域和与靶核酸杂交的导向序列；所述单链核酸检测器为含有无碱基间隔物的单链核酸检测器。
另一方面，本发明还提供了一种用于检测样品中靶核酸的试剂盒，所述试剂盒包括V型CRISPR/CAS效应蛋白、gRNA(指导RNA)和单链核酸检测器，所述gRNA包括与所述CRISPR/CAS效应蛋白结合的区域和与靶核酸杂交的导向序列；所述单链核酸检测器为含有无碱基间隔物的单链核酸检测器。
应用
另一方面，本发明还提供了上述试剂、系统、组合物或试剂盒在检测样品中靶核酸的应用。
另一方面，本发明还提供了V型CRISPR/CAS效应蛋白在检测样品中靶核酸中的应用。
另一方面，本发明还提供了上述CRISPR/CAS效应蛋白、gRNA(指导RNA)和单链核酸检测器在检测样品中靶核酸中的应用，或者，在制备用于检测样品中靶核酸的试剂、组合物、系统或试剂盒中的应用。
如上所述的应用，所述V型CRISPR/CAS效应蛋白在与样品中的靶核酸结合或杂交后，可以切割体系中的单链核酸检测器；所述单链核酸检测器为含有无碱基间隔物的单链核酸检测器。
另一方面，本发明还提供了V型CRISPR/CAS效应蛋白在制备检测样品中靶核酸的试剂中的应用。
单链核酸检测器
本发明中，所述单链核酸检测器为含有无碱基间隔物的单链核酸检测器，所述单链核酸检测器含有至少1个任意的核苷酸(任意的核苷酸缩写为N，具体可以是A、T、C、G、I、U中任意一种或几种，所述至少1个任意的核苷酸包括1-25个任意的核苷酸，例如，1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个或25个任意的核苷酸)和至少1个任意的无碱基间隔物(任意的无碱基间隔物缩写为S，具体的可以是dSpacer(abasic furan，dS)，Spacer C3，Spacer C6，Spacer C12，Spacer9，Spacer12，Spacer18，Inverted Abasic Site(dSpacer abasic furan)和rAbasic Site(rSpacer abasic furan)中的一种或任意几种，所述至少1个任意无碱基间隔物包括1-25个无碱基间隔 物，例如，1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个或25个无碱基间隔物)。
优选的，所述单链核酸检测器含有至少1个任意核苷酸和至少1个任意无碱基间隔物。
在一种实施方式中，所述单链核酸检测器含有1个任意的核苷酸，所述1个任意的核苷酸至少一端与至少1个任意无碱基间隔物连接，如单链核酸检测器序列为NS、NSS、NSSS、SN、SSN或SSSN；
或者，所述1个任意的核苷酸的两端分别与至少1个任意无碱基间隔物连接，如单链核酸检测器序列为SNS、SSNSS、SNSS或SSNS；
优选的，所述1个任意的核苷酸的两端分别连接2个任意无碱基间隔物，单链核酸检测器序列为SSNSS。
在一种实施方式中，所述单链核酸检测器有至少2个连续的核苷酸，至少2个连续的核苷酸包括2-25个连续的核苷酸，例如，2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个连续的核苷酸，所述至少2个连续的核苷酸的一端有至少1个无碱基间隔物，例如，1-25个无碱基间隔物，例如，1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个或25个无碱基间隔物；如序列为NNS、SNN、NNSS或SSNN，优选的，所述单链核酸检测器序列为C3CT、CTC3、C6CT、CTC6、TGSS、SSTS、CTS或SCT。
或者，所述至少2个连续的核苷酸的两端分别连接有至少一个无碱基间隔物，如序列为SNNS、SSNNS、SNNSS或SSNNSS，优选的，所述单链核酸检测器序列包括SCTS、SSTGS、STGSS、SSAASS、SSACSS、SSAGSS、SSATSS、SSTASS、SSTCSS、SSTGSS、SSTTSS、SSCASS、SSCCSS、SSCGSS、SSCTSS、SSGASS、SSGCSS、SSGGSS和SSGTSS中的一种或多种。
在一种实施方式中，所述单链核酸检测器含有至少2个不连续核苷酸，所述至少2个不连续核苷酸之间含有至少一个无碱基间隔物，如序列至少包括NSN、NSSN或NSSSN中的一种或多种，优选的，如序列如NSN、NSSN或NSSSN，优选的，所述单链核酸检测器序列为ASST、TSSA、ASSA、TSSST、TSST或TST。所述至少1个无碱基间隔物包括1-25个无碱基间隔物，例如，1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个或25个无碱基间隔物。
在一个具体的实施方式中，所述单链核酸检测器为含有无碱基间隔物的单链核酸检测器，所述单链核酸检测器含有至少1个任意的核苷酸和至少1个无碱基间隔物；优选的，所述单链核酸检测器不包括连续的任意两个核苷酸。上述“不包括连续的任意两个核苷酸”可以理解为，两个临近的核苷酸通过至少1个无碱 基间隔物连接，例如，1-20个无碱基间隔物；又如，2个、3个、4个、5个、6个、7个、8个、9个、10个、15个或20个无碱基间隔物。进一步的，所述单链核酸检测器中任意的单个核苷酸的两端分别连接有至少1个无碱基间隔物；更优选的，所述任意的单个核苷酸的两端分别连接有至少2个无碱基间隔物。在优选的实施方式中，所述单链核酸检测器仅含有1个任意的核苷酸和至少1个无碱基间隔物；优选的，所述核苷酸的两端分别连接有至少1个无碱基间隔物；更优选的，所述核苷酸的两端分别连接有至少2个无碱基间隔物。在一个实施方式中，所述核苷酸的两端分别连接有2-20个无碱基间隔物；优选，2个、3个、4个、5个、6个、7个、8个、9个、10个、15个或20个无碱基间隔物。在其他的实施方式中，所述单链核酸检测器具有1-300个任意核苷酸和1-300个无碱基间隔物，优选，1-200个核苷酸和1-200个无碱基间隔物，优选，1-100个核苷酸和1-100个无碱基间隔物，优选，具有1-30个核苷酸和1-30个无碱基间隔物，更优选，1个核苷酸和2-15个无碱基间隔物。
在另一个具体实施方式中，所述单链核酸检测器含有连续的2个任意核苷酸和至少2个无碱基间隔物，所述连续的2个任意核苷酸的两端分别连接有至少1个无碱基间隔物。进一步的，所述单链核酸检测器中，所述连续的2个任意核苷酸的两端分别连接有至少2个无碱基间隔物。在一个实施方式中，所述连续的2个任意核苷酸的两端分别连接有2-20个无碱基间隔物；优选，2个、3个、4个、5个、6个、7个、8个、9个、10个、15个或20个无碱基间隔物。在其他的实施方式中，所述单链核酸检测器还含有至少1组额外的核苷酸，所述每组额外的核苷酸的两端分别连接有至少1个无碱基间隔物，所述额外的核苷酸为1个任意核苷酸或连续的2个任意核苷酸。在优选的实施方式中，所述单链核酸检测器由连续的2个任意核苷酸和无碱基间隔物组成，所述连续的2个任意核苷酸的两端分别连接有2个无碱基间隔物。进一步的，所述单链核酸检测器中的核苷酸为核糖核苷酸和/或脱氧核糖核苷酸；所述核糖核苷酸的碱基选自A、U、C、G中的一种或任意几种；所述脱氧核糖核苷酸的碱基选自A、T、C、G中的一种或任意几种。在优选的实施方式中，所述连续的2个任意核苷酸选自AA、AT、AC、AG、TT、TA、TC、TG、CC、CA、CT、CG、GG、GA、GT、GC、UU、UA、UC、UG、AU、CU或GU；其中，A、C、G可以为核糖核苷酸，也可以为脱氧核糖核苷酸。在其他的实施方式中，所述单链核酸检测器具有2-300个任意核苷酸和2-300个无碱基间隔物，优选，2-200个核苷酸和2-200个无碱基间隔物，优选，2-100个核苷酸和2-100个无碱基间隔物，优选，具有2-30个核苷酸和1-30个无碱基间隔物，更优选，2个核苷酸和2-15个无碱基间隔物。
本发明中，所述无碱基间隔物选自dSpacer(abasic furan)，Spacer C3(C3)，Spacer C6(C6)，Spacer C12，Spacer9，Spacer12，Spacer18，Inverted Abasic Site(dSpacer abasic furan)和rAbasic Site(rSpacer abasic furan)中的一种或任意几种。
在一个优选的实施方式中，所述无碱基间隔物为dSpacer、Spacer C3(C3) 或Spacer C6(C6)中的一种或多种。
在一个优选的实施方式中，所述无碱基间隔物为dSpacer。所述“dSpacer”，又称为无碱基位点，四氢呋喃(tetrahydrofuran，THF)或无嘌呤/无嘧啶位点(apurinic/apyrimidinic(AP)site)，其中亚甲基位于2'-脱氧核糖的1位。dSpacer不仅在结构上与天然位点非常相似，而且相当稳定。结构如下所示：
Figure PCTCN2021105398-appb-000001
所述Spacer C3(C3)结构如下图所示：
Figure PCTCN2021105398-appb-000002
所述Spacer C6(C6)结构如下图所示：
Figure PCTCN2021105398-appb-000003
进一步的，所述核苷酸为核糖核苷酸和/或脱氧核糖核苷酸；所述核糖核苷酸的碱基选自A、U、C、G、T、I中的一种或任意几种；所述脱氧核糖核苷酸的碱基选自A、T、C、G、U、I中的一种或任意几种。
本发明中，所述单链核酸检测器不与所述gRNA杂交。
CRISPR/CAS效应蛋白
进一步的，所述V型CRISPR/CAS效应蛋白选自Cas12、Cas14家族蛋白或其突变体；在一个实施方式中，所述Cas蛋白优选为Cas12家族，包括但不限于Cas12a、Cas12b、Cas12i、Cas12j中的一种或任意几种；所述Cas14家族蛋白选自Cas14a和/或Cas14b。
在一个实施方式中，所述Cas12a选自FnCas12a、AsCas12a、LbCas12a、Lb5Cas12a、HkCas12a、OsCas12a、TsCas12a、BbCas12a、BoCas12a或Lb4Cas12a中一种或任意几种；所述Cas12a优选为LbCas12a，氨基酸序列如SEQ ID No.1所示，或者，将SEQ ID No.1所示氨基酸序或其活性片段经过一个或多个(如2个、3个、4个，5个，6个，7个，8个，9个或10个)氨基酸残基的取代、缺失或添加而形成的，且具有基本相同功能的衍生蛋白。
在其他的实施方式中，所述Cas12b的氨基酸序列如SEQ ID No.2所示，或 者，将SEQ ID No.2所示氨基酸序或其活性片段经过一个或多个(如2个、3个、4个，5个，6个，7个，8个，9个或10个)氨基酸残基的取代、缺失或添加而形成的，且具有基本相同功能的衍生蛋白。
在其他的实施方式中，所述Cas12i的氨基酸序列如SEQ ID No.3所示，或者，将SEQ ID No.3所示氨基酸序或其活性片段经过一个或多个(如2个、3个、4个，5个，6个，7个，8个，9个或10个)氨基酸残基的取代、缺失或添加而形成的，且具有基本相同功能的衍生蛋白。
在其他的实施方式中，所述Cas12j的氨基酸序列如SEQ ID No.4所示，或者，将SEQ ID No.4所示氨基酸序或其活性片段经过一个或多个(如2个、3个、4个，5个，6个，7个，8个，9个或10个)氨基酸残基的取代、缺失或添加而形成的，且具有基本相同功能的衍生蛋白。
在一个实施方式中，所述Cas蛋白突变体包括氨基酸取代、缺失或替换，且所述突变体至少保留其trans切割活性。优选地，所述突变体具有Cis和trans切割活性。
可检测信号
本发明中，所述可检测信号通过以下方式实现：基于视觉的检测，基于传感器的检测，颜色检测，基于金纳米颗粒的检测，荧光偏振，荧光信号的检测，胶体相变/分散，电化学检测和基于半导体的检测。
在一些实施方式中，本发明的方法还包括测量CRISPR/CAS效应蛋白(Cas蛋白)产生的可检测信号的步骤。所述Cas蛋白识别所述靶核酸或与所述靶核酸杂交之后可以激发单链核酸的切割活性，从而切割所述单链核酸检测器进而产生可检测信号。
本发明中，所述可检测信号可以是当切割单链核酸检测器时产生的任何信号。例如，基于金纳米颗粒的检测，荧光偏振，荧光信号的检测，胶体相变/分散，电化学检测，基于半导体的传感。所述可检测信号可通过任何合适的方式读出，包括但不限于：可检测的荧光信号的测量，凝胶电泳检测(通过检测凝胶上的条带的变化)，基于视觉或传感器的颜色的存在或不存在的检测、或者颜色存在的差异(例如，基于金纳米颗粒)以及电信号的差异。
在优选的实施方式中，所述可检测信号通过以下方式实现：所述单链核酸检测器的5’端和3’端分别设置不同的报告基团，当所述单链核酸检测器被切割后，可以表现出可检测的报告信号；例如，单链核酸检测器的两端分别设置荧光基团和淬灭基团，当所述单链核酸检测器被切割后，可以表现出可检测的荧光信号。
在一个实施方式中，所述荧光基团选自FAM、FITC、VIC、JOE、TET、CY3、CY5、ROX、Texas Red或LC RED460中的一种或任意几种；所述淬灭基团选自BHQ1、BHQ2、BHQ3、Dabcy1或Tamra中的一种或任意几种。
在其他的实施方式中，所述可检测信号还可以通过以下方式实现：所述单链核酸检测器的5’端和3’端分别设置不同的标记分子，通过胶体金检测的方式检测反应信号。
靶核酸
本发明中，所述靶核酸包括核糖核苷酸或脱氧核糖核苷酸，包括单链核酸和双链核酸，例如单链DNA、双链DNA、单链RNA、双链RNA。
在一个实施方式中，所述的靶核酸包括DNA、RNA，优选为单链核酸或双链核酸或核酸修饰物。
在一个实施方式中，所述靶核酸来源于病毒、细菌、微生物、土壤、水源、人体、动物、植物等样品。优选的，所述靶核酸为PCR、NASBA、RPA、SDA、LAMP、HAD、NEAR、MDA、RCA、LCR、RAM等方法富集或扩增的产物。
在一个实施方式中，所述方法还包括从样品中获得靶核酸的步骤。
在一个实施方式中，所述靶核酸为病毒核酸、细菌核酸、与疾病相关的特异核酸，如特定的突变位点或SNP位点或与对照有差异的核酸；优选地，所述病毒为植物病毒或动物病毒，例如，乳头瘤病毒，肝DNA病毒，疱疹病毒，腺病毒，痘病毒，细小病毒，冠状病毒；优选地，所述病毒为冠状病毒，优选地，SARS、SARS-CoV2(COVID-19)、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、Mers-Cov。
在一些实施方式中，所述靶核酸来源于细胞，例如，来源于细胞裂解液。
在一些实施方式中，所述可检测信号的测量可以是定量的，在其他的实施方式中，所述可检测信号的测量可以是定性的。
优选的，所述单链核酸检测器在被所述Cas蛋白切割之前产生第一可检测信号，并且在被切割之后产生不同于第一可检测信号的第二可检测信号。
本发明中，所述gRNA包括靶向所述靶核酸的序列(导向序列)和识别Cas蛋白的序列(同向重复序列或其部分)。
本发明中，所述的导向序列包括10-40bp；优选地，12-25bp；优选地，15-23bp；优选地，16-18bp。
比例
在一个实施方式中，所述Cas蛋白与gRNA的用量摩尔比为(0.8-1.2)：1。
在一个实施方式中，所述Cas蛋白的用量终浓度为20-200nM，优选，30-100nM，更优选，40-80nM，更优选，50nM。
在一个实施方式中，所述gRNA的用量终浓度为20-200nM，优选，30-100nM，更优选，40-80nM，更优选，50nM。
在一个实施方式中，所述靶核酸的用量终浓度为5-100nM，优选，10-50nM。
在一个实施方式中，所述单链核酸检测器的用量终浓度为100-1000nM，优选，150-800nM，优选，200-800nM，优选，200-500nM，优选，200-300nM。
一般定义：
术语“杂交”或“互补的”或“基本上互补的”是指核酸(例如RNA、DNA)包含使其能够非共价结合的核苷酸序列，即以序列特异性，反平行的方式(即核酸特异性结合互补核酸)与另一核酸形成碱基对和/或G/U碱基对，“退火”或“杂交”。杂交需要两个核酸含有互补序列，尽管碱基之间可能存在错配。两个核酸之间杂交的合适条件取决于核酸的长度和互补程度，这是本领域公知的变量。典型地，可杂交核酸的长度为8个核苷酸或更多(例如，10个核苷酸或更多，12个核苷酸或更多，15个核苷酸或更多，20个核苷酸或更多，22个核苷酸或更多，25个核苷酸或更多，或30个核苷酸或更多)。
应当理解，多核苷酸的序列不需要与其靶核酸的序列100％互补以特异性杂交。多核苷酸可包含60％或更高，65％或更高，70％或更高，75％或更高，80％或更高，85％或更高，90％或更高，95％或更高，98％或更高，99％或更高，99.5％或更高，或与其杂交的靶核酸序列中的靶区域的序列互补性为100％。
除非另有定义，否则本文所用的技术和科学术语具有与所属领域的普通技术人员之一通常理解的相同的含义。
术语“氨基酸”是指含有氨基的羧酸。生物体内的各种蛋白质是由20种基本氨基酸构成的。
术语“多核苷酸”、“核苷酸序列”、“核酸序列”、“核酸分子”和“核酸”可以互换使用，包括DNA、RNA或者其杂交体，可以是双链或单链的。
术语“同源性”或“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，氨基酸序列的同一性可以通过常规方法，参考例如Smith and Waterman,1981,Adv.Appl.Math.2:482Pearson&Lipman,1988,Proc.Natl.Acad.Sci.USA 85:2444，Thompsonetal.,1994,Nucleic Acids Res 22:467380等的教导，通过计算机化运行运算法则(Wisconsin Genetics软件包中的GAP,BESTFIT,FASTA,和TFASTA，Genetics Computer Group)来确定。也可使用可从美国国立生物技术信息中心(NCBI www.ncbi.nlm.nih.gov/)获得的BLAST运算法则，使用默认参数确定。
如本文所用，所述“CRISPR”是指成簇、规律间隔的短回文重复序列(Clustered regularly interspaced short palindromic repeats)，其来自微生物的免疫系统。
如本文所用，“生物素(biotin)”也称维生素H，是一种分子量为244Da的小分子维生素。“亲和素(avidin)”，又称抗生物素，是一种碱性糖蛋白，具有4个同生物素亲和例极高的结合位点，常用亲和素有链霉亲合素。生物素与亲和素的极强亲和力可用于在检测体系中放大或增强检测信号。如生物素很易与蛋白质 (如抗体等)以共价键结合，而结合了酶的亲和素分子与结合有特异性抗体的生物素分子产生反应，既起到了多级放大作用，又由于酶在遇到相应底物时的催化作用而呈色，达到检测未知抗原(或抗体)分子的目的。
单链核酸检测器
本发明所述的单链核酸检测器在检测方法或系统中用以报告是否含有靶序列。所述的单链核酸检测器两端包括不同的报告基团或标记分子，当其处于初始状态(即未被切割状态时)不呈现报告信号，当该单链核酸检测器被切割后，呈现出可检测的信号，即切割后与切割前表现出可检测的区别。在本发明中，如果能够检测出可检测的区别，则反映靶核酸中含有待检测的靶序列；或者，如果无法检检测出所述的可检测的区别，则反映靶核酸中不含有待检测的靶序列。
在一个实施方式中，所述的报告基团或标记分子包括荧光基团和淬灭基团，所述荧光基团选自FAM、FITC、VIC、JOE、TET、CY3、CY5、ROX、Texas Red或LC RED460中的一种或任意几种；所述淬灭基团选自BHQ1、BHQ2、BHQ3、Dabcy1或Tamra中的一种或任意几种。
在一个实施方式中，所述的单链核酸检测器具有连接至5’端第一分子(如FAM或FITC)和连接至3’端的第二分子(如生物素)。所述的含有单链核酸检测器的反应体系与流动条配合用以检测特征序列(优选，胶体金检测方式)。所述的流动条被设计为具有两条捕获线，在样品接触端(胶体金)设有结合第一分子的抗体(即第一分子抗体)，在第一线(control line)处含有结合第一分子抗体的抗体，在第二线(test line)处含有与第二分子结合的第二分子的抗体(即第二分子抗体，如亲和素)。当反应沿着条带流动时，第一分子抗体与第一分子结合携带切割或未切割的寡核苷酸至捕获线，切割的报告子将在第一个捕获线处结合第一分子抗体的抗体，而未切割的报告子将在第二捕获线处结合第二分子抗体。报告基团在各条线的结合将导致强读出/信号(例如颜色)。随着更多的报告子被切割，更多的信号将在第一捕获线处累积，并且在第二线处将出现更少的信号。
在某些方面，本发明涉及如本文所述的流动条用于检测核酸的用途。
在某些方面，本发明涉及用本文定义的流动条检测核酸的方法，例如(侧)流测试或(侧)流免疫色谱测定。
在某些方面，所述单链核酸检测器中的分子可相互替换，或改变分子的位置，只要其报告原理与本发明相同或相近，所改进的方式也均包含在本发明中。
本发明所述的检测方法，可用于待检测特征序列的定量检测。所述的定量检测指标可以根据报告基团的信号强弱进行定量，如根据荧光基团的发光强度，或根据显色条带的宽度等。
无碱基间隔物
如本文所用，“无碱基间隔物(Spacer，在本发明中缩写为S)”是指表示不包含具体编码信息的核苷。无碱基间隔物可与寡核苷酸结合，包括3’和5’末端，或核苷酸链内部。常见的Spacer包括：dSpacer(abasic furan)，Spacer C3，Spacer C6，Spacer C12，Spacer 9，Spacer12，Spacer18，Inverted Abasic Site(dSpacer abasic furan)和rAbasic Site(rSpacer abasic furan)。
上述无碱基间隔物是本领域公知的无碱基间隔物，例如，美国专利US8153772B2中公开了dSpacer，Spacer 9，Spacer 18，Spacer C3；中国专利申请CN101454451A公开了dSpacer。
本文优选的无碱基间隔物“dSpacer”又称为无碱基位点，，在本发明中缩写为dS，四氢呋喃(tetrahydrofuran，THF)或无嘌呤/无嘧啶位点(apurinic/apyrimidinic(AP)site)，或，无碱基连接子，其中亚甲基位于2'-脱氧核糖的1位。dSpacer不仅在结构上与天然位点非常相似，而且相当稳定。结构如下所示：
Figure PCTCN2021105398-appb-000004
所述dSpacer在与核苷酸连接时，可以形成如下的结构：
Figure PCTCN2021105398-appb-000005
Cas蛋白
本文所述“Cas蛋白”是指CRISPR-associated蛋白，优选来自V型或VI型CRISPR/CAS蛋白，其一旦与待检测特征序列(靶序列)结合(即形成Cas蛋白-gRNA-靶序列的三元复合物)，就可以诱发其trans活性，即随机切割非靶向 单链核苷酸(即本文所述单链核酸检测器)。当Cas蛋白与特征序列结合后，其切割或不切割特征序列，均可以诱发其trans活性；优选地，其通过切割特征序列诱发其trans活性；更优选地，其通过切割单链特征序列诱发其trans活性。所述Cas蛋白通过识别与特征序列临近的PAM(protospacer adjacent motif)识别特征序列。
本发明所述的Cas蛋白为至少具有trans切割活性的蛋白，优选地，所述的Cas蛋白为具有Cis和trans切割活性的蛋白。所述的Cis活性是指Cas蛋白可在gRNA的作用下识别PAM位点并特异性切割靶序列的活性。
本发明所述的Cas蛋白包括V型CRISPR/CAS效应蛋白，包括Cas12、Cas14等蛋白家族。优选地，例如Cas12蛋白，例如Cas12a、Cas12b、Cas12i、Cas12j；优选地，所述Cas蛋白为Cas12a、Cas12b、Cas12i、Cas12j；Cas14蛋白家族包括Cas14a、Cas14b等。
在实施方式中，本文所称的Cas蛋白，如Cas12，也涵盖Cas的功能变体或其同源物或直系同源物。如本文所用的蛋白的“功能变体”是指至少部分保留该蛋白的活性的这样的蛋白的变体。功能变体可以包括突变体(其可以是插入、缺失或替换突变体)，包括多晶型物等。功能变体中还包括这样的蛋白与另一种通常不相关的核酸、蛋白质、多肽或肽的融合产物。功能变体可以是天然存在的或可以是人造的。有利的实施方式可以涉及工程化或非天然存在的V型DNA靶向效应蛋白。
在一个实施方式中，编码Cas蛋白，如Cas12，的一种或多种核酸分子或其直系同源物或同源物可以被密码子优化用于在真核细胞中表达。真核生物可如本文所述。一种或多种核酸分子可以是工程化的或非天然存在的。
在一个实施方式中，Cas12蛋白或其直系同源物或同源物可以包含一个或多个突变(并且因此编码其的核酸分子可以具有一个或多个突变。突变可以是人工引入的突变并且可以包括但不限于催化结构域中的一个或多个突变。
在一个实施方式中，Cas蛋白可以来自：纤毛菌属、李斯特菌属、棒状杆菌属、萨特氏菌属、军团菌属、密螺旋体属、产线菌属、真细菌属、链球菌属、乳酸菌属、支原体属、拟杆菌属、Flaviivola、黄杆菌属、固氮螺菌属、Sphaerochaeta、葡糖醋杆菌属、奈瑟氏菌属、罗氏菌属、Parvibaculum、葡萄球菌属、Nitratifractor、支原体属、弯曲杆菌属和毛螺菌属。
在一个实施方式中，Cas蛋白选自如下序列组成的蛋白：
(1)SEQ ID No.1-4所示的蛋白；
(2)将SEQ ID No.1-4所示氨基酸序或其活性片段列经过一个或多个(如2个、3个、4个，5个，6个，7个，8个，9个或10个)氨基酸残基的取代、缺失或添加而形成的，且具有基本相同功能的衍生蛋白。
在一个实施方式中，所述Cas蛋白还包括与上述序列具有50％，优选55％，优选60％，优选65％，优选70％，优选75％，优选80％，优选85％，优选90％，优选95％，序列同一性的，且具有trans活性的蛋白。
所述的Cas蛋白可以通过重组表达载体技术获得，即将编码该蛋白的核酸分子构建到合适的载体上，再转化到宿主细胞中，使得所述的编码核酸分子在细胞中表达，从而获得相应的蛋白。所述的蛋白可以被细胞分泌出来，或者破解细胞通过常规的提取技术获得该蛋白。所述的编码核酸分子可以整合至宿主细胞的基因组中进行表达，也可以不整合到宿主细胞中进行表达。所述的载体还进一步包括有利于序列整合，或进行自我复制的调节元件。所述的载体可以是例如质粒、病毒、粘粒、噬菌体等类型，它们是本领域技术人员所熟知的，优选地，本发明中的表达载体是质粒。所述的载体进一步包括一种或多种调控元件，选自启动子、增强子、翻译起始的核糖体结合位点、终止子、多聚腺苷酸序列、筛选标记基因。
宿主细胞可以是原核细胞，如大肠杆菌，链霉菌属、农杆菌：或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物细胞。本领域一般技术人员都清楚如何选择适当的载体和宿主细胞。
gRNA
如本文所用，所述的“gRNA”又称为guide RNA或导向RNA，并且具有本领域技术人员通常理解的含义。一般而言，导向RNA可以包含同向(direct)重复序列和导向序列(guide sequence)，或者基本上由或由同向重复序列和导向序列(在内源性CRISPR系统背景下也称为间隔序列(spacer))组成。gRNA在不同的CRISPR系统中，依据其所依赖的Cas蛋白的不同，可以包括crRNA和tracrRNA，也可以只含有crRNA。crRNA和tracrRNA可以经过人工改造融合形成single guide RNA(sgRNA)。在某些情况下，导向序列是与靶序列(本发明中所述特征序列)具有足够互补性从而与所述靶序列杂交并引导CRISPR/Cas复合物与所述靶序列的特异性结合的任何多核苷酸序列，通常具有12-25nt的序列长度。所述的同向重复序列可折叠形成特定结构(如茎环结构)供Cas蛋白识别，以形成复合物。所述的导向序列不需要与特征序列(靶序列)100％互补。所述的导向序列不与单链核酸检测器互补。
在某些实施方案中，当最佳比对时，导向序列与其相应靶序列之间的互补程度(匹配度)为至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、或至少99％。确定最佳比对在本领域的普通技术人员的能力范围内。例如，存在公开和可商购的比对算法和程序，诸如但不限于ClustalW、matlab中的史密斯-沃特曼算法(Smith-Waterman)、Bowtie、Geneious、Biopython以及SeqMan。
本发明所述的gRNA可以是天然的，也可以是经过人工改造或设计合成的。
靶核酸
如本文所用，所述“靶核酸”是指从生物样品(待测样品)中提取的多核苷酸分子。所述生物样品是从任何生物体获得、排泄或分泌的任何固体或流体样品，包括但不限于单细胞生物，例如细菌、酵母、原生动物和变形虫等，多细胞生物 (例如植物或动物，包括来自健康或表面健康的人类受试者或受待诊断或调查的病症或疾病影响的人类患者的样品，例如病原微生物例如病原细菌或病毒的感染)。例如，生物样品可以是从例如血液、血浆、血清、尿液、粪便、痰液、粘液、淋巴液、滑液、胆汁、腹水、胸腔积液、血清肿、唾液、脑脊液、水性或玻璃体液、或任何身体分泌物、渗出液、渗出液(例如，从脓肿或任何其他感染或炎症部位获得的液体)中获得的生物液体或从关节(例如，正常关节或受疾病影响的关节，例如类风湿性关节炎、骨关节炎、痛风或脓毒性关节炎)获得的液体，或皮肤或粘膜表面的拭子。样品也可以是从任何器官或组织获得的样品(包括活组织检查或尸体解剖标本，例如肿瘤活检)或者可以包含细胞(原代细胞或培养的细胞)或由任何细胞、组织或器官调理的培养基。示例性的样品包括但不限于，细胞、细胞裂解物、血涂片、细胞离心制剂、细胞学涂片、体液(例如血液、血浆、血清、唾液、痰、尿、支气管肺泡灌洗、精液等)、组织活检(例如肿瘤活组织检查)、细针抽吸物和/或组织切片(例如低温恒温器组织切片和/或石蜡包埋的组织切片)。
在其他实施方式中，生物样品可以是植物细胞、愈伤、组织或器官(如根、茎、叶、花、种子、果实)等。
本发明中，所述的靶核酸还包括通过逆转录RNA形成的DNA分子，进一步地，所述的靶核酸可以采用本领域公知的技术对其进行扩增，所述的扩增技术等温扩增技术和非等温扩增技术，等温扩增可以是基于核酸测序的扩增(NASBA)、重组酶聚合酶扩增(RPA)、环介导的等温扩增(LAMP)、链置换扩增(SDA)、解旋酶依赖性扩增(HDA)、或切口酶扩增反应(NEAR)。在某些示例性实施方式中，可以使用非等温扩增方法，其包括但不限于PCR、多重置换扩增(MDA)、滚环扩增(RCA)、连接酶链反应(LCR)、或衍生物扩增方法(RAM)。
进一步的，本发明所述的检测方法还一步包括对靶核酸扩增的步骤；所述的检测系统，还进一步包括对靶核酸进行扩增的试剂。所述扩增的试剂包括下组中的一种或多种：DNA聚合酶、链置换酶、解旋酶、重组酶、单链结合蛋白等。
附图说明
图1.当探针序列为5’6-FAM/S//S//A//S//S//3’BHQ1时，Cas12a、Cas12j和Cas12b能够快速的报告出荧光。
图2.当探针序列为5’6-FAM/S//S//T//S//S//3’BHQ1时，Cas12a、Cas12j和Cas12b能够快速的报告出荧光。
图3.当探针序列为5’6-FAM/S//S//G//S//S//3’BHQ1时，Cas12a、Cas12j和Cas12b能够快速的报告出荧光。
图4.当探针序列为5’6-FAM/S//S//C//S//S//3’BHQ1时，Cas12a、Cas12j和Cas12b能够快速的报告出荧光。
图5.当探针序列为5’6-FAM/S//S//N//S//S//3’BHQ1时，Cas12i能够快速的报告出荧光。
图6.当探针序列为5’6-FAM//T//T//3’BHQ1时，Cas12a和Cas12b几乎 不能报告出荧光，Cas12i可以报告出荧光信号。
图7.当探针序列为5’6-FAM//T//G//3’BHQ1时，Cas12a和Cas12b几乎不能报告出荧光，Cas12i可以报告出荧光信号。
图8.当探针序列为5’6-FAM//C//A//3’BHQ1时，Cas12a、Cas12i和Cas12b均不能报告出明显荧光信号。
图9.当探针序列为5’6-FAM//C//T//3’BHQ1时，Cas12a和Cas12b几乎不能报告出荧光，Cas12i可以报告出荧光信号。
图10.当探针序列为5’6-FAM//S//S//A//A//S//S//3’BHQ1时,Cas12a、Cas12b和Cas12i均能报告出明显荧光信号。
图11.当探针序列为5’6-FAM//S//S//A//C//S//S//3’BHQ1时,Cas12a、Cas12b和Cas12i均能报告出明显荧光信号。
图12.当探针序列为5’6-FAM//S//S//A//G//S//S//3’BHQ1时,Cas12a、Cas12b和Cas12i均能报告出明显荧光信号。
图13.当探针序列为5’6-FAM//S//S//A//T//S//S//3’BHQ1时,Cas12a、Cas12b和Cas12i均能报告出明显荧光信号。
图14.当探针序列为5’6-FAM//S//S//C//A//S//S//3’BHQ1时,Cas12a、Cas12b和Cas12i均能报告出明显荧光信号。
图15.当探针序列为5’6-FAM//S//S//C//C//S//S//3’BHQ1时,Cas12a、Cas12b和Cas12i均能报告出明显荧光信号。
图16.当探针序列为5’6-FAM//S//S//C//G//S//S//3’BHQ1时,Cas12a、Cas12b和Cas12i均能报告出明显荧光信号。
图17.当探针序列为5’6-FAM//S//S//C//T//S//S//3’BHQ1时,Cas12a、Cas12b和Cas12i均能报告出明显荧光信号。
图18.当探针序列为5’6-FAM//S//S//G//A//S//S//3’BHQ1时,Cas12a、Cas12b和Cas12i均能报告出明显荧光信号。
图19.当探针序列为5’6-FAM//S//S//G//C//S//S//3’BHQ1时,Cas12a、Cas12b和Cas12i均能报告出明显荧光信号。
图20.当探针序列为5’6-FAM//S//S//G//G//S//S//3’BHQ1时,Cas12a、Cas12b和Cas12i均能报告出明显荧光信号。
图21.当探针序列为5’6-FAM//S//S//G//T//S//S//3’BHQ1时,Cas12a、Cas12b和Cas12i均能报告出明显荧光信号。
图22.当探针序列为5’6-FAM//S//S//T//A//S//S//3’BHQ1时,Cas12a、Cas12b和Cas12i均能报告出明显荧光信号。
图23.当探针序列为5’6-FAM//S//S//T//C//S//S//3’BHQ1时,Cas12a、Cas12b和Cas12i均能报告出明显荧光信号。
图24.当探针序列为5’6-FAM//S//S//T//G//S//S//3’BHQ1时,Cas12a、Cas12b和Cas12i均能报告出明显荧光信号。
图25.当探针序列为5’6-FAM//S//S//T//T//S//S//3’BHQ1时,Cas12a、 Cas12b和Cas12i均能报告出明显荧光信号。
图26.当探针序列为SCTS时,Cas12a、Cas12b、Cas12i和Cas12j均能报告出明显荧光信号。
图27.当探针序列为SSTGS时,Cas12a、Cas12b、Cas12i和Cas12j均能报告出明显荧光信号。
图28.当探针序列为C3CT时,Cas12a、Cas12b、Cas12i和Cas12j均能报告出明显荧光信号。
图29.当探针序列为CTC3时,Cas12a、Cas12b、Cas12i和Cas12j均能报告出明显荧光信号。
图30.当探针序列为C6CT时,Cas12a、Cas12b、Cas12i和Cas12j均能报告出明显荧光信号。
图31.当探针序列为CTC6时,Cas12a、Cas12b、Cas12i和Cas12j均能报告出明显荧光信号。
图32.当探针序列为TGSS时,Cas12a、Cas12b、Cas12i和Cas12j均能报告出明显荧光信号。
图33.当探针序列为SCT时,Cas12a、Cas12b、Cas12i和Cas12j均能报告出明显荧光信号。
图34.当探针序列为TST时,Cas12a、Cas12b、Cas12i和Cas12j均能报告出明显荧光信号。
图35.当探针序列为TSST时,Cas12a、Cas12b、Cas12i和Cas12j均能报告出明显荧光信号。
图36.当探针序列为TSSST时,Cas12a、Cas12b、Cas12i和Cas12j均能报告出明显荧光信号。
图37.当探针序列为TSSA时,Cas12a、Cas12b、Cas12i和Cas12j均能报告出明显荧光信号。
图38.当探针序列为ASST时,Cas12a、Cas12b、Cas12i和Cas12j均能报告出明显荧光信号。
图39.当探针序列为ASSA时,Cas12a、Cas12b、Cas12i和Cas12j均能报告出明显荧光信号。
实施方式
下面结合实施例对本发明做进一步的说明，以下所述，仅是对本发明的较佳实施例而已，并非对本发明做其他形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容，依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化，均落在本发明的保护范围内。
本发明技术方案基于如下原理，获得待测样品的核酸，比如，可以通过扩增的方法得到靶核酸，利用可以与靶核酸配对的gRNA引导Cas蛋白识别并结合在靶核酸上；随后，Cas蛋白激发单链核酸检测器的切割活性，从而切割体系里的 单链核酸检测器；单链核酸检测器的两端分别设置荧光基团和淬灭基团，如果单链核酸检测器被切割，则会激发荧光；如果单链核酸检测器无法被切割，则不会激发荧光；在其他的实施方式中，单链核酸检测器的两端还可以设置成能够被胶体金检测的标记。
实施例1、利用含有无碱基间隔物的核酸检测器进行靶核酸的检测(SSNSS)
本实施方式中，利用结构为dSpacer-dSpacer-N-dSpacer-dSpacer的含有无碱基间隔物的核酸检测器作为检测探针，其中，N为任意的脱氧核糖核苷酸，dSpacer缩写为dS。
所述含有无碱基间隔物的核酸检测器探针序列为5’6-FAM/dS//dS//A//dS//dS//3’BHQ1，5’6-FAM/dS//dS//T//dS//dS//3’BHQ1，5’6-FAM/dS//dS//C//dS//dS//3’BHQ1或5’6-FAM/dS//dS//G//dS//dS//3’BHQ1；所述无碱基间隔物选用为dSpacer，所述A、T、C、G为DNA。
申请人对Cas12a(SEQ ID No.1)、Cas12b(SEQ ID No.2)和Cas12j(SEQ ID No.4)，在利用上述含有无碱基间隔物的核酸检测器时的检测效果进行了验证，试验设计如下：
Figure PCTCN2021105398-appb-000006
上述Cas12i3-g2-ssDNA0的序列如SEQ ID No.5所示；
上述Cas12j19-g3-ssDNA0的序列如SEQ ID No.6所示；
上述LbCas12a-TGW6-g1的序列如SEQ ID No.7所示；
上述AaCas12b-TGW6-g1的序列如SEQ ID No.8所示；
上述Cas12j19-TGW6-g3的序列如SEQ ID No.9所示。
20微升体系内各成分的含量如下：
组分 20ul体系使用量 终浓度
缓冲液 2ul 1×
2uM的Cas12 0.5ul 50nM
1uM的gRNA 1ul 50nM
100nM的ssDNA 1ul 5nM
10uM的单链核酸检测器 0.4ul 200nM
H 2O Up to 20ul  
当探针序列为5’6-FAM/dS//dS//A//dS//dS//3’BHQ1时，结果如图1所示，Cas12a、Cas12j和Cas12b能够快速的报告出荧光。
当探针序列为5’6-FAM/dS//dS//T//dS//dS//3’BHQ1时，结果如图2所示，Cas12a、Cas12j和Cas12b能够快速的报告出荧光。
当探针序列为5’6-FAM/dS//dS//G//dS//dS//3’BHQ1时，结果如图3所示，Cas12a、Cas12j和Cas12b能够快速的报告出荧光。
当探针序列为5’6-FAM/dS//dS//C//dS//dS//3’BHQ1时，结果如图4所示，Cas12a、Cas12j和Cas12b能够快速的报告出荧光。
实施例2、当Cas效应蛋白选用Cas12i时，利用含有无碱基间隔物的核酸检测器进行靶核酸的检测(SSNSS)
选用序列为5’6-FAM/dS//dS//N//dS//dS//3’BHQ1的单链核酸检测器，所述无碱基间隔物选用为dSpacer，按照实施例1的实验体系对Cas12i(SEQ ID No.3所示，某些情况下，也用Cas12i3来指代)进行验证，靶核酸为Cas12i3-g2-ssDNA0，gRNA为DRi3-gOsTGW6-2(序列为agagaaugugugcauagucacacuuucaccgacagcagcaugaacu)。
实验结果如图5所示，Cas12i识别到靶核酸后激活的旁路激活活性可以切割序列为5’6-FAM/dS//dS//N//dS//dS//3’BHQ1(无碱基间隔物选用为dSpacer)的单链核酸检测器，也就是Cas12i也可以切割含有无碱基间隔物的单链核酸检测器。
实施例3、对比两个连续任意核苷酸和两端连接有无碱基间隔物的连续两个任意核苷酸对检测效果的影响(NN对比SSNNSS)
本实施方式中，利用序列为连续的2个任意核苷酸的核酸检测器进行靶核酸的检测，具体的连续的2个任意核苷酸分别是TT、TG、CA和CT；上述序列的两端分别连接有淬灭基团和荧光基团。
本实施方式中，还利用结构为5’6-FAM//S//S//N//N//S//S//3’BHQ1的含有无碱基间隔物的核酸检测器作为检测探针，其中无碱基间隔物选用为dSpacer(dS)，N//N为连续的2个任意核苷酸，具体的N//N的序列为AA、AT、AC、AG、TA、TT、TC、TC、CA、CT、CC、CG、GA、GT、GC和GG，所述A、T、C、G为DNA。
申请人以上述序列为NN和SSNNSS的共20个单链核酸检测器，对Cas12a(SEQ ID No.1)、Cas12b(SEQ ID No.2)和Cas12i(SEQ ID No.3)，按照实施例1中Cas蛋白对应的靶核酸和gRNA用20微升体系进行了验证。
当探针序列为NN时，实验结果如图6-9所示，Cas12a和Cas12b几乎不能报告出荧光，Cas12i可以报告出荧光信号。
当探针序列为SSNNSS时，实验结果如图10-25所示，Cas12a(SEQ ID No.1)、Cas12b(SEQ ID No.2)和Cas12i(SEQ ID No.3)均可以切割结构为 5’6-FAM//dS//dS//N//N//dS//dS//3’BHQ1的含有无碱基间隔物的核酸检测器，并能报告出荧光。
实施例4、对比两个连续任意核苷酸两端连接的无碱基间隔物的数量的不同对检测效果的影响(SNNS、SNNSS、SSNNS)
在验证了在两个连续任意核苷酸两端分别连接两个无碱基间隔物可以显著提高检测效果后，对两端连接的无碱基间隔物的个数进行了调整，具体的，合成序列为dS//C//T//dS、dS//dS//T//G//dS的单链核酸检测器(无碱基间隔物选用为dSpacer，两端分别连接有淬灭基团和荧光基团)，依然按照前述实施例中的体系进行检测。
实验结果如图26和图27所示，单链核酸检测器序列为dS//C//T//dS、dS//dS//T//G//dS时，Cas12a(SEQ ID No.1)、Cas12b(SEQ ID No.2)、Cas12i(SEQ ID No.3)和Cas12j(SEQ ID No.4)均可以报告出荧光信号。实验证明，两个连续任意核苷酸两端连接无碱基间隔物，可以提高检测效率，与无碱基间隔物数目无关。
实施例5、两个连续任意核苷酸只有一端(3’端或5’端)连接无碱基间隔物对检测效果的影响(NNS、NNSS、SNN、SSNN)
按照前述实施例中的实验方法，合成不同序列的核苷酸和不同无碱基间隔物组合的单链核酸检测器进行验证，实验证明含有无碱基间隔物的单链核酸检测器可以被Cas效应蛋白的旁路切割活性切割，显示出荧光信号。
实验结果如图28-33所示，单链核酸检测器的序列为C3CT、CTC3、C6CT、CTC6、T//G//dSpacer//dSpacer、dSpacer//C//T时，Cas12a(SEQ ID No.1)、Cas12b(SEQ ID No.2)、Cas12i(SEQ ID No.3)和Cas12j(SEQ ID No.4)均可以报告出荧光信号。
实施例6、不连续的任意核苷酸之间连接不同数量的无碱基间隔物，对检测效果的影响
当单链核酸检测器中含有两个或以上的任意核苷酸，且核苷酸之间包含有无碱基间隔物时，也可以作为单链核酸检测器；并且不受核苷酸之间无碱基间隔物的数目影响。
按照前述实验方法进行操作，实验结果如图34-39所示，当单链核酸检测器的序列为T//dS//T、T//dS//dS//T、T//dS//dS//dS//T、A//dS//dS//A、A//dS//dS//T、A//dS//dS//A时，Cas12a(SEQ ID No.1)、Cas12b(SEQ ID No.2)、Cas12i(SEQ ID No.3)和Cas12j(SEQ ID No.4)均可以报告出荧光信号。

Claims (10)

  1. 一种检测样品中靶核酸的方法，所述方法包括将样品与V型Cas蛋白(CRISPR/CAS效应蛋白)、gRNA(指导RNA)和单链核酸检测器接触，所述gRNA包括与所述V型Cas蛋白结合的区域和与靶核酸杂交的导向序列；检测由V型Cas蛋白切割单链核酸检测器产生的可检测信号，从而检测靶核酸；所述单链核酸检测器含有至少1个任意的核苷酸和至少1个任意的无碱基间隔物；所述单链核酸检测器不与所述gRNA杂交。
  2. 根据权利要求1所述的方法，其特征在于，所述无碱基间隔物选自dSpacer，Spacer C3(C3)，Spacer C6(C6)，Spacer C12，Spacer9，Spacer12，Spacer18，Inverted Abasic Site(dSpacer abasic furan)和rAbasic Site(rSpacer abasic furan)中的一种或任意几种；优选的，所述无碱基间隔物为dSpacer、C3和C6中的一种或任意几种。
  3. 根据权利要求1或2所述的方法，其特征在于，所述单链核酸检测器选自以下(1)-(3)中的一种或任意几种：
    (1)所述单链核酸检测器含有1个任意的核苷酸，所述1个任意的核苷酸的至少一端与至少1个任意无碱基间隔物连接，优选的，所述1个任意的核苷酸的两端分别与至少1个任意无碱基间隔物连接，更优选的，所述1个任意的核苷酸的两端分别连接2个任意无碱基间隔物；
    (2)所述单链核酸检测器含有至少2个连续的核苷酸，所述至少2个连续的核苷酸的一端连接有至少一个无碱基间隔物，优选的，所述至少2个连续的核苷酸的两端分别连接有至少一个无碱基间隔物；
    (3)所述单链核酸检测器含有至少2个不连续核苷酸，所述不连续核苷酸之间含有至少一个无碱基间隔物。
  4. 如权利要求1-3任一所述的方法，其特征在于，所述V型Cas蛋白选自Cas12、Cas14家族蛋白的任意一种或任意几种组合；优选的，所述Cas14家族蛋白选自Cas14a、Cas14b中的一种或任意几种组合；更优选的，所述Cas12家族蛋白为Cas12i、Cas12j、Cas12a、Cas12b中的一种或任意几种组合物。
  5. 根据权利要求1所述的方法，其特征在于，所述可检测信号通过以下方式进行检测：基于视觉的检测，基于传感器的检测，颜色检测，基于金纳米颗粒的检测，荧光偏振，荧光信号的检测，胶体相变/分散，电化学检测和基于半导体的检测。
  6. 根据权利要求1所述的方法，其特征在于，所述靶核酸包括核糖核苷酸或脱氧核糖核苷酸；优选的，包括单链核酸、双链核酸，例如，单链DNA、双链DNA、单链RNA。
  7. 根据权利要求3所述的方法，其特征在于，所述单链核酸检测器的5’端和3’端分别设置不同的报告基团，当所述单链核酸检测器被切割后，可以表现出可检测的报告信号；或者，所述单链核酸检测器的5’端和3’端分别设置不同的标记分子，通过胶体金检测的方式检测反应信号。
  8. 根据权利要求1所述的方法，其特征在于，所述靶核酸来源于病毒、细 菌、微生物、土壤、水源、人体、动物、植物等样品；优选的，所述靶核酸为病毒核酸、细菌核酸、与疾病相关的特异核酸或与对照有差异的特异核酸，优选地，疾病相关的特异核酸为特定的突变位点或SNP位点；优选地，所述病毒为植物病毒或动物病毒，例如，乳头瘤病毒，肝DNA病毒，疱疹病毒，腺病毒，痘病毒，细小病毒，冠状病毒；优选地，所述病毒为冠状病毒，例如，SARS、SARS-CoV2(COVID-19)、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、Mers-Cov。
  9. 一种用于检测样品中靶核酸的试剂、系统、组合物或试剂盒，所述试剂、系统、组合物包括权利要求1-8任一权利要求中所述的V型Cas蛋白、gRNA和单链核酸检测器。
  10. 权利要求1-8任一权利要求中所述的V型Cas蛋白、gRNA和单链核酸检测器，或，权利要求9所述的试剂、系统、组合物或试剂盒在检测样品中靶核酸的应用。
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