WO2022042568A1 - 基于crispr技术进行多重核酸检测的方法 - Google Patents

基于crispr技术进行多重核酸检测的方法 Download PDF

Info

Publication number
WO2022042568A1
WO2022042568A1 PCT/CN2021/114374 CN2021114374W WO2022042568A1 WO 2022042568 A1 WO2022042568 A1 WO 2022042568A1 CN 2021114374 W CN2021114374 W CN 2021114374W WO 2022042568 A1 WO2022042568 A1 WO 2022042568A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
detector
target
stranded
detection composition
Prior art date
Application number
PCT/CN2021/114374
Other languages
English (en)
French (fr)
Inventor
梁亚峰
孙洁
刘锐恒
Original Assignee
山东舜丰生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 山东舜丰生物科技有限公司 filed Critical 山东舜丰生物科技有限公司
Priority to US17/648,180 priority Critical patent/US20220136075A1/en
Publication of WO2022042568A1 publication Critical patent/WO2022042568A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • 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
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
    • C12Q2521/307—Single strand endonuclease
    • 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
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/113—Modifications characterised by incorporating modified backbone
    • 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
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/119—Modifications characterised by incorporating abasic sites

Definitions

  • the invention relates to the field of nucleic acid detection, to a method for multiple nucleic acid detection based on CRISPR technology, in particular to a method, system and kit for target nucleic acid detection based on CRISPR technology, and in particular to a method for multiple target nucleic acid detection based on CRISPR technology.
  • 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 present invention provides a method for nucleic acid detection based on CRISPR technology, especially a method, system and kit for multiplex detection of nucleic acid.
  • the present invention provides a method for detecting a target nucleic acid in a sample, the method comprising contacting the sample with a nucleic acid detection composition, the nucleic acid detection composition comprising a Cas protein, a gRNA and a single-stranded nucleic acid detector; the The gRNA includes a region that binds to the Cas protein and a guide sequence that hybridizes with the target sequence on the target nucleic acid; detecting the detectable signal generated by the Cas protein cleavage single-stranded nucleic acid detector, thereby detecting the target nucleic acid;
  • the nucleic acid detection composition is selected from any one, any two, any three or four of the first nucleic acid detection composition, the second nucleic acid detection composition, the third nucleic acid detection composition and the fourth nucleic acid detection composition. kind;
  • the first nucleic acid detection composition includes Cas12i, a first gRNA that can bind to Cas12i and a first target sequence hybridized to a target nucleic acid, and a first single-stranded nucleic acid detector;
  • the second nucleic acid detection composition includes Cas12b, a second gRNA that can bind to Cas12b and a second target sequence hybridized to the target nucleic acid, and a second single-stranded nucleic acid detector;
  • the third nucleic acid detection composition includes Cas12a, a third gRNA that can bind to Cas12a and a third target sequence hybridized to the target nucleic acid, and a third single-stranded nucleic acid detector;
  • the fourth nucleic acid detection composition includes Cas12j, a fourth gRNA that can bind to Cas12j and a fourth target sequence hybridized to the target nucleic acid, and a fourth single-stranded nucleic acid detector.
  • the first single-stranded nucleic acid detector contains at least two consecutive nucleotides, and the nucleotides are one or more of ribonucleotides, deoxyribonucleotides, and nucleic acid analogs; the ribose
  • the base of the nucleotide is selected from one or any of several in A, U, C, G, T, I; the base of the deoxyribonucleotide is selected from A, T, C, G, U, One or any of I.
  • the nucleic acid of the first single-stranded nucleic acid detector is two consecutive nucleotides, and the nucleotides are one or more of ribonucleotides, deoxyribonucleotides, and nucleic acid analogs .
  • the nucleic acid analogs include 2'-fluoro group modification, 2' oxymethyl modification, locked nucleic acid, bridge nucleic acid, morpholine nucleic acid, ethylene glycol nucleic acid, hexitol nucleic acid, threose nucleic acid, arabinose nucleic acid, 2' Methoxyacetyl modification, 2'-amino modification, 4'-thio RNA and combinations thereof; preferably, the nucleic acid analog is a 2'-fluoro modified nucleic acid analog.
  • the base of the ribonucleotide is selected from one or any of A, U, C, G, T, and I; the base of the deoxyribonucleotide is selected from A, T, One or any of C, G, U, and I.
  • the base of the nucleic acid analog is selected from one or any of A, U, C, G, T, and I; preferably, the base of the nucleic acid analog is selected from T and/or C.
  • the nucleic acid of the first single-stranded nucleic acid detector is two consecutive deoxynucleotides, and the base sequence of the deoxyribonucleotides is TT or CT.
  • the first single-stranded nucleic acid detector is two consecutive nucleic acid analogs.
  • the first single-stranded nucleic acid detector is two consecutive 2'-fluoro-modified nucleic acid analogs.
  • the first single-stranded nucleic acid detector is two consecutive 2'-fluoro-modified Ts, or a single-chain composed of 2'-fluoro-modified T and 2'-fluoro-modified C .
  • the second single-stranded nucleic acid detector is selected from a single-stranded nucleic acid detector containing an abasic spacer; or, the nucleic acid structure of the second single-stranded nucleic acid detector is a nucleic acid analog, and the nucleic acid analog is a lock Nucleic acid (LNA), a single-stranded nucleic acid detector comprising locked nucleic acid is also described in Chinese application CN2020105609327.
  • the bases of the locked nucleic acid are selected from one or any of A, T, C, G, U, and I.
  • the single-stranded nucleic acid detector containing an abasic spacer contains at least one arbitrary nucleotide and at least one abasic spacer; preferably, the two ends of the nucleotide are respectively connected with at least one abasic spacers; more preferably, at least two abasic spacers are connected to both ends of the nucleotides; in a preferred embodiment, the single-stranded nucleic acid detector contains only one arbitrary Nucleotides.
  • the single-stranded nucleic acid detector containing an abasic spacer comprises at least 2 discontinuous arbitrary nucleotides, and at least one non-consecutive arbitrary nucleotides are connected to each other.
  • Base spacer in one embodiment, 2-20 abasic spacers are connected between the discontinuous arbitrary nucleotides; preferably, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 15 or 20 abasic spacers.
  • 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 containing an abasic spacer contains one arbitrary nucleotide, and two abasic spacers are connected to both ends of the nucleotide, respectively. .
  • abasic spacer is selected from one or any of several in dSpacer, Spacer C3, Spacer C6, Spacer C12, Spacer9, Spacer12, Spacer18, Inverted Abasic Site (dSpacer abasic furan) and rAbasic Site (rSpacer abasic furan) ;
  • the abasic spacer is dSpacer (abasic furan).
  • dSpacer is also referred to as abasic site, tetrahydrofuran (THF) or apurine/apyrimidinic (AP) site or, abasic linker, wherein methylene
  • THF tetrahydrofuran
  • AP purine/apyrimidinic
  • base is located at the 1-position of 2'-deoxyribose.
  • dSpacers are abasic spacers well known in the art, eg dSpacers are disclosed in US Pat. No. 8,153,772B2. 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:
  • the 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 kinds; the base of the deoxyribonucleotide is selected from one or any of several of A, T, C, G, I, and U.
  • nucleotides are deoxyribonucleotides; the bases of the deoxyribonucleotides are selected from one or any of A, T, and G.
  • the third single-stranded nucleic acid detector is a single-stranded nucleic acid detector containing an abasic spacer; the single-stranded nucleic acid detector containing an abasic spacer contains at least one arbitrary nucleotide and at least one Abasic spacer; preferably, at least one abasic spacer is connected to both ends of the nucleotide; more preferably, at least two abasic spacers are connected to both ends of the nucleotide Spacer; in a preferred embodiment, the single-stranded nucleic acid detector contains only one arbitrary 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 containing an abasic spacer contains one arbitrary nucleotide, and two abasic spacers are connected to both ends of the nucleotide, respectively. .
  • the abasic spacer is dSpacer (abasic furan).
  • the 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 kinds; the base of the deoxyribonucleotide is selected from one or any of several of A, T, C, G, I, and U.
  • the fourth single-stranded nucleic acid detector is selected from a single-stranded nucleic acid detector containing an abasic spacer; or, the nucleic acid structure of the fourth single-stranded nucleic acid detector is a nucleic acid analog, and the nucleic acid analog is 2 'Oxymethyl RNA, the base of the 2' oxymethyl RNA is selected from one or any of A, T, U, C, G, and I.
  • the single-stranded nucleic acid detector containing an abasic spacer contains at least one arbitrary nucleotide and at least one abasic spacer; preferably, the two ends of the nucleotide are respectively connected with at least one abasic spacers; more preferably, at least two abasic spacers are connected to both ends of the nucleotides; in a preferred embodiment, the single-stranded nucleic acid detector contains only one arbitrary Nucleotides.
  • 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 containing an abasic spacer contains one arbitrary nucleotide, and two abasic spacers are connected to both ends of the nucleotide, respectively. .
  • the abasic spacer is dSpacer (abasic furan).
  • the 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; The bases of the deoxyribonucleotides are selected from one or any of A, T, C, G, I, and U.
  • nucleotides are deoxyribonucleotides; the base of the deoxyribonucleotides is T.
  • the Cas12i can specifically cleave the first single-stranded nucleic acid detector compared with other Cas proteins, thereby generating a first detectable signal; the Cas12b can specifically cleave compared with other Cas proteins The second single-stranded nucleic acid detector generates a second detectable signal; the Cas12a can specifically cleave the third single-stranded nucleic acid detector compared with other Cas proteins, thereby generating a third detectable signal; Compared with other Cas proteins, the Cas12j can specifically cleave the fourth single-stranded nucleic acid detector, thereby generating a fourth detectable signal.
  • the above-mentioned specific cleavage means that a certain protein, compared with other proteins, has higher cleavage efficiency and better detectable signal for the single-stranded nucleic acid detector it targets.
  • the detectable signal is achieved by vision-based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization, fluorescence signal-based detection, colloidal phase transition/dispersion, electrochemical detection, and Inspection of semiconductors.
  • 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 first detectable signal, the second detectable signal, the third detectable signal and the fourth detectable signal are different detection signals from each other.
  • the two ends of the single-stranded nucleic acid detector are respectively provided with a fluorescent group and a quenching group, and when the single-stranded nucleic acid detector is cleaved, a detectable fluorescent signal can be displayed.
  • 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 BHQ1, BHQ2, BHQ3 , one or any of Dabcy1 or Tamra.
  • both ends of the first single-stranded nucleic acid detector, the second single-stranded nucleic acid detector, the third single-stranded nucleic acid detector and the fourth single-stranded nucleic acid detector are respectively provided with a first fluorescent group,
  • the group, the second fluorophore, the third fluorophore, and the fourth fluorophore may be the same or different fluorophores; the first quenching group, the second quenching group, the third The quenching group and the fourth quenching group may be the same or different quenching groups from each other.
  • the 5' end and the 3' end of the single-stranded nucleic acid detector are respectively provided with different label molecules, and the detection of the single-stranded nucleic acid detector before and after being cleaved by Cas protein is detected by colloidal gold detection.
  • 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.
  • first target sequence second target sequence
  • third target sequence fourth target sequence
  • the above-mentioned target sequences are different target sequences from each other.
  • the method for detecting target nucleic acid of the present invention can realize multiple detection of nucleic acid in a sample; in one embodiment, the first target sequence and the second target sequence , the third target sequence and the fourth target sequence may be target sequences designed for the same target nucleic acid or different sites of the same gene, or target sequences designed for different target nucleic acids or different genes.
  • different target sequences can be designed for a certain bacterium, virus or disease-related nucleic acid; in other embodiments, different target sequences can be designed for different kinds of bacteria, virus or disease-related nucleic acid. target sequence.
  • the first nucleic acid detection composition can be used in combination with any one of the second nucleic acid detection composition, the third nucleic acid detection composition, or the fourth nucleic acid detection composition, thereby realizing dual detection of the target nucleic acid .
  • the second nucleic acid detection composition can be used in combination with any one of the third nucleic acid detection composition or the fourth nucleic acid detection composition, thereby realizing double detection of the target nucleic acid; in this implementation, the nucleic acid structure of the second single-stranded nucleic acid detector in the second nucleic acid detection composition is a nucleic acid analog, and the nucleic acid analog is a locked nucleic acid (LNA).
  • LNA locked nucleic acid
  • the second nucleic acid detection composition can be used in combination with the fourth nucleic acid detection composition to achieve dual detection of target nucleic acid; in this embodiment, the second nucleic acid detection composition
  • the second single-stranded nucleic acid detector in the composition is selected from single-stranded nucleic acid detectors containing an abasic spacer, preferably, the nucleotides in the second single-stranded nucleic acid detector in the second nucleic acid detection composition are
  • the base is selected from one or more of A, T, and G;
  • the nucleic acid structure of the fourth single-stranded nucleic acid detector is a nucleic acid analog, and the nucleic acid analog is 2' oxymethyl RNA; preferably, the The base of the 2' oxymethyl RNA is selected from one or any of several of A, T, U, C, G, and I.
  • the third nucleic acid detection composition can be used in combination with the fourth nucleic acid detection composition, thereby realizing double detection of target nucleic acid; in this embodiment, the third nucleic acid detection composition
  • the third single-stranded nucleic acid detector in is selected from the single-stranded nucleic acid detector containing an abasic spacer; the nucleic acid structure of the fourth single-stranded nucleic acid detector is a nucleic acid analog, and the nucleic acid analog is a 2' oxygen Methyl RNA; preferably, the base of the 2' oxymethyl RNA is selected from one or any of A, T, U, C, G, and I.
  • the first nucleic acid detection composition, the second nucleic acid detection composition, and any one composition selected from the third nucleic acid detection composition and the fourth nucleic acid detection composition can be used in combination, thereby achieving Triple detection of target nucleic acid;
  • the nucleic acid structure of the second single-stranded nucleic acid detector in the second nucleic acid detection composition is a nucleic acid analog, and the nucleic acid analog is a locked nucleic acid (LNA) .
  • LNA locked nucleic acid
  • the third nucleic acid detection composition, the fourth nucleic acid detection composition, and any one composition selected from the first nucleic acid detection composition and the second nucleic acid detection composition can be used in combination, so as to achieve Triple detection of target nucleic acid;
  • the nucleic acid structure of the second single-stranded nucleic acid detector in the second nucleic acid detection composition is a nucleic acid analog, and the nucleic acid analog is a locked nucleic acid (LNA)
  • LNA locked nucleic acid
  • the base of the nucleotide in the third single-stranded nucleic acid detector in the third nucleic acid detection composition is C;
  • the nucleic acid structure of the fourth single-stranded nucleic acid detector is a nucleic acid analog, and the nucleic acid is similar
  • the substance is 2' oxymethyl RNA, and the base of the 2' oxymethyl RNA is selected from one or any of several A, T, U, C, G, and I.
  • the first nucleic acid detection composition, the second nucleic acid detection composition, the third nucleic acid detection composition and the fourth nucleic acid detection composition can be used in combination, thereby realizing quadruple detection of the target nucleic acid;
  • the nucleic acid structure of the second single-stranded nucleic acid detector in the second nucleic acid detection composition is a nucleic acid analog, and the nucleic acid analog is a locked nucleic acid (LNA);
  • LNA locked nucleic acid
  • the base of the nucleotide in the third single-stranded nucleic acid detector in the third nucleic acid detection composition is C;
  • the nucleic acid structure of the fourth single-stranded nucleic acid detector is a nucleic acid analog, and the nucleic acid analog is 2' oxymethyl RNA, and the base of the 2' oxymethyl RNA is selected from one or any of several A, T, U, C, G, and I.
  • the first nucleic acid detection composition and the second nucleic acid detection composition are used for dual detection
  • different target sequences can be designed for the SARS-CoV2 (COVID-19) virus
  • two target sequences for the SARS-CoV2 (COVID-19) virus can be designed Dual detection of target nucleic acid
  • the first target sequence and the second target sequence can be designed for SARS-CoV2 (COVID-19) and SARS virus, respectively, so as to perform dual detection of SARS-CoV2 (COVID-19) and SARS virus detection.
  • the present invention provides a method for multiplex detection of target nucleic acid in a sample, the method comprising contacting the sample with 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 includes a region that binds to the Cas protein and a guide sequence that hybridizes with a target sequence on the target nucleic acid; detects a detectable signal generated by the Cas protein cleavage single-stranded nucleic acid detector, thereby detecting the target nucleic acid;
  • the nucleic acid detection The composition is selected from any one, any two, any 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.
  • the present invention provides a nucleic acid detection composition selected from the above-mentioned first nucleic acid detection composition, second nucleic acid detection composition, third nucleic acid detection composition and fourth nucleic acid detection composition Any one, any two, any three or four of the compositions.
  • the present invention also provides a system for detecting target nucleic acid in a sample
  • the system includes a nucleic acid detection composition
  • the nucleic acid detection composition includes a Cas protein, a gRNA and a single-stranded nucleic acid detector
  • the The gRNA includes a region bound to the Cas protein and a guide sequence that hybridizes with the target sequence on the target nucleic acid
  • the nucleic acid detection composition is selected from the above-mentioned first nucleic acid detection composition, second nucleic acid detection composition, third nucleic acid detection composition Any one, any two, any three or four of the detection composition and the fourth nucleic acid detection composition.
  • the present invention also provides a kit for detecting target nucleic acid in a sample
  • the kit includes a nucleic acid detection composition
  • the nucleic acid detection composition includes a Cas protein, a gRNA and a single-stranded nucleic acid detector
  • the gRNA includes a region that binds to the Cas protein and a targeting sequence that hybridizes to a target sequence on a target nucleic acid.
  • the nucleic acid detection composition is selected from any one, any two, or any three 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 or four.
  • the present invention also provides the application of the above system or kit in detecting target nucleic acid in a sample.
  • the system or kit of the present invention detects the target nucleic acid in the sample
  • the first nucleic acid detection composition, the second nucleic acid detection composition, the third nucleic acid detection composition and the fourth nucleic acid detection composition can be used
  • One or any of several kinds of detection can be used to detect the same target sequence, or to detect different target sequences, so that double, triple or quadruple detection effect can be achieved.
  • the present invention also provides the use of the nucleic acid detection composition in detecting target nucleic acid in a sample, or in preparing a system or kit for detecting target nucleic acid in a sample.
  • the nucleic acid detection composition is selected from any one, any two, or any three 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 or four.
  • 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 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 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 of the present invention 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 method further comprises the step of obtaining the target nucleic acid from the sample.
  • 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 guide sequence includes 10-40bp; preferably, 12-25bp; preferably, 15-23bp; preferably, 16-18bp.
  • the gRNA has at least 50% matching degree 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%.
  • the characteristic sites are completely matched to the gRNA.
  • one or more gRNAs with different targeting sequences may be included in the detection method, which target different target sequences.
  • 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.
  • 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.
  • oligonucleotide refers to a sequence containing 3-100 nucleotides, preferably, 3-30 nucleotides, preferably, 4-20 nucleotides, more preferably, 5-15 nucleotides acid.
  • 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.
  • nucleic acid analogs include, but are not limited to: 2' oxymethyl ( -OCH3 ) RNA, locked nucleic acid, bridge nucleic acid, morpholine nucleic acid, glycol nucleic acid, hexitol nucleic acid, threose nucleic acid, Arabinucleic acid, 2'methoxyacetyl RNA, 2'-fluoro(-F)RNA, 2'-amino RNA, 4'-thio RNA, and combinations thereof, including optional ribonucleotides or deoxyribonuclei nucleotide residues.
  • LNAs Locked Nucleic Acids
  • LNAs are 2' modified nucleosides that contain a diradical linking the C2' and C4' of the ribose ring of the nucleoside that constrains or locks the conformation of the ribose ring, its structural formula As shown below, the bases of LNA can be selected from adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil.
  • 2'O-Methyl RNA (2'O-Methyl RNA, 2'O-methyl, 2'-O-methyl substituted RNA, -OCH 3 ):
  • 2'O-Methyl RNA is a 2'-modified nucleoside that A methoxy group ( -OCH3 ) is attached at the C2' position of the ribose sugar ring of the nucleoside.
  • the 2' oxymethyl RNA monomer structure is shown below, the bases of 2' oxymethyl RNA can be selected from adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil.
  • 2'-Fluoro-modified nucleic acid analogs are 2'-modified nucleosides to which an F(-F) is attached at the C2' position of the ribose sugar ring of the nucleoside .
  • the monomer structure of 2'-fluoroRNA is shown below, the base of 2'-fluoroRNA can be selected from adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil.
  • Spacer 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, Spacer9, 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 known as an abasic site, tetrahydrofuran (THF) or apurine/apyrimidinic (AP) site, or, abasic linker sub, in which the methylene group is located at the 1-position of 2'-deoxyribose.
  • dSpacer is not only structurally very similar to the natural site, but also quite stable. The structure looks like this:
  • the dSpacer can form the following structure when the nucleotides are connected:
  • 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.
  • 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, ordinarily 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 carrier, 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 usually 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.
  • the two ends of the single-stranded nucleic acid detector of the present invention include different reporter groups or labeling molecules, and when the single-stranded nucleic acid detector is in an initial state (ie, a non-cleaved state), no report signal is presented.
  • a detectable signal is present, that is, a detectable difference is exhibited after cleavage and before cleavage.
  • the detectable difference can be detected, it reflects that the target nucleic acid contains the characteristic sequence to be detected; or, if the detectable difference cannot be detected, it reflects that the target nucleic acid does not contain the to be detected. feature 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).
  • 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.
  • Cas12b can be specific It is a cleavage single-stranded nucleic acid detector with better detectable signal than other proteins.
  • Cas12a and Cas12b can specifically cleave single-stranded nucleic acid detectors when the single-stranded nucleic acid detector sequence is 5'6-FAM/S//S//A//S//S//3'BHQ1 , showed better detectable signal than other proteins, and the detectable signal of Cas12a was stronger than that of Cas12b.
  • FIG. 8 Double detection of N and S genes of COVID-19 virus using Cas12i and Cas12j: cas12i targets S gene, reporter is FAM-CT-BHQ1, cas12j targets N gene, and reporter is Cy3-SSTSS-BHQ2; When both S gene and N gene are present in the sample, two fluorescent signals can be detected; when the N gene but not the S gene is present in the sample, only the FAM fluorescent signal corresponding to Cas12i can be detected; the presence of the S gene in the sample does not In the presence of the N gene, only the Cy3 fluorescence signal corresponding to Cas12j can be detected; in the absence of the S gene or the N gene in the sample, neither signal can be detected.
  • Figure 9 Triple detection of different target nucleic acids using Cas12a, Cas12b, Cas12i.
  • 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; 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.
  • different single-stranded nucleic acid detectors are designed, and Cas12i, Cas12j, Cas12a and Cas12b are used for detection.
  • Different single-stranded nucleic acid detectors are, single-stranded nucleic acid detector-TT, single-stranded nucleic acid detector-TT-F, single-stranded nucleic acid detector-LNA, single-stranded nucleic acid detector-SSCSS, single-stranded nucleic acid detector- SSASS, Single Stranded Nucleic Acid Detector-SSTSS, Single Stranded Nucleic Acid Detector-SSGSS and Single Stranded Nucleic Acid Detector- OCH3 .
  • the structure of single-stranded nucleic acid detector-TT is 5'6-FAM//T//T//3'BHQ1
  • the structure of single-stranded nucleic acid detector-TT-F is 5'6-FAM//TF// TF//3'BHQ1 (wherein, TF is T modified by 2' fluorine group)
  • the structure of single-stranded nucleic acid detector-LNA is 5'6-FAM//LNA_T//LNA_T//LNA_T//LNA_T //LNA_T//3'BHQ1
  • the structure of single-stranded nucleic acid detector-SSCSS is 5'6-FAM//S//S/C//S//S//3'BHQ1, where S is dSpacer
  • the structure of single-stranded nucleic acid detector-SSASS is 5'6-FAM//S//S/A//S//S//3'BHQ1
  • S is d
  • Cas12a SEQ ID No. 1
  • Cas12b SEQ ID No. 2
  • Cas12i SEQ ID No. 3
  • Cas12j SEQ ID No. 4
  • 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:
  • Cas12i can specifically cleave the single-stranded nucleic acid detector, which shows better detectable signal than other proteins.
  • Cas12i can specifically cleave the single-stranded nucleic acid detector, which shows better detectable signal than other proteins.
  • Cas12i can also specifically cut the single-stranded nucleic acid detector, showing produce a better detectable signal.
  • Cas12b can specifically cleave the single-stranded nucleic acid detector, which shows better detectable signal than other proteins.
  • Cas12a can specifically cut the single-stranded nucleic acid detector, which is better than other proteins detectable signal.
  • Cas12a and Cas12b can specifically cleave single-stranded nucleic acid detectors, which are better than other proteins. Better detectable signal.
  • Cas12a and Cas12j can specifically cleave single-stranded nucleic acid detectors, which are better than other proteins. Better detectable signal.
  • Cas12a and Cas12b can specifically cleave single-stranded nucleic acid detectors, which are better than other proteins. Better detectable signal, the detectable signal of Cas12b is stronger than that of Cas12a.
  • Cas12j can specifically cleave the single-stranded nucleic acid detector, which shows a better detectable signal than other proteins.
  • cas12i targets the S gene
  • the reporter is 5'6-FAM//C//T//3'BHQ1
  • the gRNA sequence is AGAGAAUGUGUGCAUAGUCACACUCAGGAUGUUAACUGCACAG, the same SEQ ID No.11
  • cas12j targets the N gene
  • the reporter is 5'Cy3//S//S//T//S//S//3'BHQ2
  • the gRNA sequence is GUGCUGCUGUCUCCCAGACGGGAGGCAGAACUGCACCGCGACAUUCCGAAGAACGC, the same as SEQ ID No. 12 shown.
  • cas12a targets EV71 VP1 target nucleic acid, its sequence is GTGCACGCAACAAAAGTGAACTCTGCATCAAAGCGCATGT, and the single-stranded nucleic acid detector is 5'6-FAM//A//dS//dS//T//3'BHQ1 (where dSpacer is an abasic spacer ), the gRNA is LbCas12a-g71-1, and its sequence is UAAUUUCUACUAAGUGUAGAU AUGCAGAGUUCACUUUGUUGCG (bold is the position where the gRNA is combined with the protein, and the underlined part is the position matching the target nucleic acid sequence);
  • cas12b targets OsTGW6 target nucleic acid
  • its sequence is GATCGTTGGTAGTTCATGCTGCTGTCGGTGAAATAAACATCTCCGGTAAC
  • the single-stranded nucleic acid detector is 5'TAMRA//LNA-T//LNA-T//LNA-T//LNA-T//LNA-T//3' BHQ2
  • LNA-T refers to a locked nucleic acid with a base of T
  • the tracrRNA sequence is GUCUAAAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGCCCGUUGAACUUCUCAAAAAGAACGCUCGCUCAGUGUUCUGAC
  • the crRNA sequence is GUCGGAUCACUGAGCGAGCGAUCUGAGAAGUGGCAC uuucaccgacagcagcauga (the underlined part is the position that matches the target nucleic acid sequence);
  • cas12i targets the COVID-19 orf1ab target nucleic acid, its sequence is Ggcaccaaattccaaaggtttaccttggtaatcatcttcagtaccatactcatattgag, the single-stranded nucleic acid detector is 5'HEX//C//T//3'BHQ1, the gRNA is CV19-Lamb-i3g5g, and its sequence is AGAGAAUGUGUGCAUAGUCACAC ccaaggUaaaccUUUggaaUUUgg ( The bold is the position where the gRNA binds to the protein, and the underlined part is the position that matches the target nucleic acid sequence).
  • the left side of the figure refers to the target nucleic acid added in the system, which is represented by the abbreviation of the corresponding enzyme.
  • “ABI” refers to the EV71 VP1 target nucleic acid, Cas12b (Cas12b) detected by adding Cas12a (A) protein to the system.
  • the fluorescent signal generated by the stranded nucleic acid detector, such as "Cas12-FAM” means that after the Cas12 protein in this system recognizes the EV71 VP1 target nucleic acid, the bypass cleavage activity is activated, and the specific cleavage of the single stranded nucleic acid detector 5' 6-FAM//A//S//S//T//3'BHQ1, the FAM fluorescence intensity produced, the darker the color, the stronger the signal.
  • the OsTGW6 target nucleic acid detected by Cas12b(B) protein and the COVID-19 orf1ab detected by Cas12i(I) protein are added to the system
  • FAM fluorescence corresponding to Cas12a, TAMRA fluorescence corresponding to Cas12b and HEX fluorescence corresponding to Cas12i can be detected.
  • the detection results prove that Cas12a, Cas12b and Cas12i have different preferences for single-stranded nucleic acid detectors and can be used for triple nucleic acid detection.

Abstract

本发明提供一种基于CRISPR技术检测靶核酸的方法、系统和试剂盒，所述方法包括将样品与核酸检测组合物接触，所述核酸检测组合物包括Cas蛋白、gRNA和单链核酸检测器；所述gRNA包括与所述Cas蛋白结合的区域和与靶核酸上的靶序列杂交的导向序列；检测由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。

Description

基于CRISPR技术进行多重核酸检测的方法 技术领域
本发明涉及核酸检测领域，涉及基于CRISPR技术进行多重核酸检测的方法，具体涉及基于CRISPR技术进行靶核酸检测的方法、系统和试剂盒，尤其涉及一种基于CRISPR技术进行多重靶核酸检测的方法。
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。
尽管现有核酸检测技术众多，如何更加快速、简便、廉价、精确的检测仍然是改进检测技术的重要方向，尤其是如何对核酸进行多重检测，是亟待解决的问题。
发明内容
本发明提供了一种基于CRISPR技术进行核酸检测的方法，尤其是对核酸进行多重检测的方法、系统和试剂盒。
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核酸检测组合物接触，所述核酸检测组合物包括Cas蛋白、gRNA和单链核酸检测器；所述gRNA包括与所述Cas蛋白结合的区域和与靶核酸上的靶序列杂交的导向序列；检测由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；
所述核酸检测组合物选自第一核酸检测组合物、第二核酸检测组合物、第三核酸检测组合物和第四核酸检测组合物中的任意一种、任意两种、任意三种或四种；
所述第一核酸检测组合物包括Cas12i，可以结合Cas12i以及与靶核酸上的第一靶序列杂交的第一gRNA，和第一单链核酸检测器；
所述第二核酸检测组合物包括Cas12b，可以结合Cas12b以及与靶核酸上的第二靶序列杂交的第二gRNA，和第二单链核酸检测器；
所述第三核酸检测组合物包括Cas12a，可以结合Cas12a以及与靶核酸上的第三靶序列杂交的第三gRNA，和第三单链核酸检测器；
所述第四核酸检测组合物包括Cas12j，可以结合Cas12j以及与靶核酸上的第四靶序列杂交的第四gRNA，和第四单链核酸检测器。
所述第一单链核酸检测器含有至少两个连续的核苷酸，所述核苷酸为核糖核苷酸、脱氧核糖核苷酸、核酸类似物中的一种或几种；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。
优选的，所述第一单链核酸检测器的核酸为两个连续的核苷酸，所述核苷酸为核糖核苷酸、脱氧核糖核苷酸、核酸类似物中的一种或几种。
所述核酸类似物包括2’-氟基修饰、2’氧甲基修饰、锁核酸、桥核酸、吗啉核酸、乙二醇核酸、己糖醇核酸、苏糖核酸、阿拉伯糖核酸、2’甲氧基乙酰基修饰、2’-氨基修饰、4’-硫RNA及其组合；优选的，所述核酸类似物为2’-氟基修饰的核酸类似物。
进一步的，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中的一个或任意几种。所述核酸类似物的碱基选自A、U、C、G、T、I中的一种或任意几种；优选的，所述核酸类似物的碱基选自T和/或C。
优选的，所述第一单链核酸检测器的核酸为两个连续的脱氧核苷酸，所述脱氧核糖核苷酸的碱基序列为TT或CT。
优选的，所述第一单链核酸检测器为两个连续的核酸类似物。
更优选的，所述第一单链核酸检测器为两个连续的2’-氟基修饰的核酸类似物。
进一步的，所述第一单链核酸检测器为两个连续的2’-氟基修饰的T，或者是，2’-氟基修饰的T和2’-氟基修饰的C组成的单链。
所述第二单链核酸检测器选自含有无碱基间隔物的单链核酸检测器；或者，所述第二单链核酸检测器的核酸结构为核酸类似物，所述核酸类似物为锁 核酸(LNA)，包含锁核酸的单链核酸检测器还记载在了中国申请CN2020105609327中。所述锁核酸的碱基选自A、T、C、G、U、I中的一种或任意几种。
所述含有无碱基间隔物的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。
在一个实施方式中，所述含有无碱基间隔物的单链核酸检测器包括至少2个不连续的任意的核苷酸，所述不连续的任意的核苷酸之间连接至少1个无碱基间隔物；在一个实施方式中，所述不连续的任意的核苷酸之间连接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。
在最优选的实施方式中，所述含有无碱基间隔物的单链核酸检测器含有1个任意的核苷酸，在所述核苷酸的两端分别连接有2个无碱基间隔物。
所述无碱基间隔物选自dSpacer，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；优选的，所述无碱基间隔物为dSpacer(abasic furan)。
本发明中，“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)或，无碱基连接子，其中亚甲基位于2'-脱氧核糖的1位。
dSpacer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。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 PCTCN2021114374-appb-000001
所述dSpacer在与核苷酸连接时，可以形成如下的结构：
Figure PCTCN2021114374-appb-000002
优选的，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、I、U中的一种或任意几种。
进一步的，所述核苷酸为脱氧核糖核苷酸；所述脱氧核糖核苷酸的碱基选自A、T、G中的一种或任意几种。
所述第三单链核酸检测器是含有无碱基间隔物的单链核酸检测器；所述含有无碱基间隔物的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。
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。
在最优选的实施方式中，所述含有无碱基间隔物的单链核酸检测器含有1个任意的核苷酸，在所述核苷酸的两端分别连接有2个无碱基间隔物。
所述无碱基间隔物为dSpacer(abasic furan)。
优选的，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、I、U中的一种或任意几种。
所述第四单链核酸检测器选自含有无碱基间隔物的单链核酸检测器；或者，所述第四单链核酸检测器的核酸结构为核酸类似物，所述核酸类似物为2’氧甲基RNA，所述2’氧甲基RNA的碱基选自A、T、U、C、G、I中的一种或任意几种。
所述含有无碱基间隔物的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。
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。
在最优选的实施方式中，所述含有无碱基间隔物的单链核酸检测器含有1个任意的核苷酸，在所述核苷酸的两端分别连接有2个无碱基间隔物。
所述无碱基间隔物为dSpacer(abasic furan)。
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、I、U中的一种或任意几种。
进一步的，所述核苷酸为脱氧核糖核苷酸；所述脱氧核糖核苷酸的碱基为T。
本发明中，所述Cas12i与其他Cas蛋白相比可以特异性的切割所述第一单链核酸检测器，从而产生第一可检测信号；所述Cas12b与其他Cas蛋白相比可以特异性的切割所述第二单链核酸检测器，从而产生第二可检测信号；所述Cas12a与其他Cas蛋白相比可以特异性的切割所述第三单链核酸检测器，从而产生第三可检测信号；所述Cas12j与其他Cas蛋白相比可以特异性的切割所述第四单链核酸检测器，从而产生第四可检测信号。
上述特异性的切割，是指某个蛋白与其他蛋白相比，对于其针对的单链核酸检测器，切割效率更高，所表现出的可检测信号更好。
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，基于荧光信号的检测，胶体相变/分散，电化学检测和基于半导体的检测。
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，基于荧光信号的检测，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，第一可检测信号、第二可检测信号、第三可检测信号和第四可检测信号彼此之间是不同的检测信号。
优选地，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。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，所述第一单链核酸检测器，第二单链核酸检测器，第三单链核酸检测器和第四单链核酸检测器的两端分别设置第一荧光基团、第一淬灭基团，第二荧光基团、第二淬灭基团，第三荧光基团、第三淬灭基团，第四荧光基团、第四淬灭基团；上述第一荧光基团、第二荧光基团、第三荧光基团、第四荧光基团可以是彼此之间相同或不同的荧光基团；上述第一淬灭基团、第二淬灭基团、第三淬灭基团、第四淬灭基团可以是彼此之间相同或不同的淬灭基团。
在其他的实施方式中，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，检测所述单链核酸检测器被Cas蛋白切割前和被Cas蛋白切割后的胶体金测试结果；所述单链核酸检测器被Cas蛋白切割前和被Cas蛋白切割后在胶体金的检测线和质控线上将表现出不同的显色结果。
本发明中，所述第一靶序列、第二靶序列、第三靶序列、第四靶序列可以是相同的靶序列，或者，彼此之间是不同的靶序列。
本领域技术人员根据实际需要可以选择上述第一靶序列、第二靶序列、第三靶序列、第四靶序列是相同的，或者是不同的，或者是部分相同的。
优选的，上述靶序列是彼此之间不同的靶序列，如此设置，本发明检测靶核酸的方法可以实现对于样品中核酸的多重检测；在一个实施方式中，第一靶序列、第二靶序列、第三靶序列、第四靶序列可以是针对同一靶核酸或同一基因的不同位点设计的靶序列，或者是，针对不同靶核酸或不同基因设计的靶序列。在一个实施方式中，可以针对某一种细菌、病毒或疾病相关的核酸，设计不同的靶序列；在其他的实施方式中，可以是针对不同种类的细菌、病毒或疾病相关的核酸，设计不同的靶序列。
在一个实施方式中，可以采用第一核酸检测组合物与第二核酸检测组合物、第三核酸检测组合物或第四核酸检测组合物的任意一种组合使用，从而实现对靶核酸的双重检测。
在另一种实施方式中，可以采用第二核酸检测组合物与第三核酸检测组合物或第四核酸检测组合物的任意一种组合使用，从而实现对靶核酸的双重检测；在这种实施方式中，所述第二核酸检测组合物中的第二单链核酸检测器的核酸结构为核酸类似物，所述核酸类似物为锁核酸(LNA)。
在另一种实施方式中，可以采用第二核酸检测组合物与第四核酸检测组合物组合使用，从而实现对靶核酸的双重检测；在这种实施方式中，所述第二核酸检测组合物中的第二单链核酸检测器选自含有无碱基间隔物的单链核酸检测器，优选的，所述第二核酸检测组合物中的第二单链核酸检测器中的核苷酸的碱基选自A、T、G中的一个或几个；所述第四单链核酸检测器的核酸结构为 核酸类似物，所述核酸类似物为2’氧甲基RNA；优选的，所述2’氧甲基RNA的碱基选自A、T、U、C、G、I中的一种或任意几种。
在另一种实施方式中，可以采用第三核酸检测组合物与第四核酸检测组合物组合使用，从而实现对靶核酸的双重检测；在这种实施方式中，所述第三核酸检测组合物中的第三单链核酸检测器选自含有无碱基间隔物的单链核酸检测器；所述第四单链核酸检测器的核酸结构为核酸类似物，所述核酸类似物为2’氧甲基RNA；优选的，所述2’氧甲基RNA的碱基选自A、T、U、C、G、I中的一种或任意几种。
在另一种实施方式中，可以采用第一核酸检测组合物、第二核酸检测组合物以及选自第三核酸检测组合物和第四核酸检测组合物的任意一种组合物联用，从而实现对靶核酸的三重检测；在这种实施方式中，所述第二核酸检测组合物中的第二单链核酸检测器的核酸结构为核酸类似物，所述核酸类似物为锁核酸(LNA)。
在另一种实施方式中，可以采用第三核酸检测组合物、第四核酸检测组合物以及选自第一核酸检测组合物和第二核酸检测组合物的任意一种组合物联合使用，从而实现对靶核酸的三重检测；在这种实施方式中，所述第二核酸检测组合物中的第二单链核酸检测器的核酸结构为核酸类似物，所述核酸类似物为锁核酸(LNA)；所述第三核酸检测组合物中的第三单链核酸检测器中的核苷酸的碱基为C；所述第四单链核酸检测器的核酸结构为核酸类似物，所述核酸类似物为2’氧甲基RNA，所述2’氧甲基RNA的碱基选自A、T、U、C、G、I中的一种或任意几种。
在其他的实施方式中，可以采用第一核酸检测组合物、第二核酸检测组合物、第三核酸检测组合物和第四核酸检测组合物组合使用，从而实现对靶核酸的四重检测；在这种实施方式中，所述第二核酸检测组合物中的第二单链核酸检测器的核酸结构为核酸类似物，所述核酸类似物为锁核酸(LNA)；在这种实施方式中，所述第三核酸检测组合物中的第三单链核酸检测器中的核苷酸的碱基为C；所述第四单链核酸检测器的核酸结构为核酸类似物，所述核酸类似物为2’氧甲基RNA，所述2’氧甲基RNA的碱基选自A、T、U、C、G、I中的一种或任意几种。
例如，采用第一核酸检测组合物和第二核酸检测组合物进行双重检测时，可以针对SARS-CoV2(COVID-19)病毒设计不同的靶序列，对SARS-CoV2(COVID-19)的两个靶核酸进行双重检测；或者，可以针对SARS-CoV2(COVID-19)和SARS病毒分别设计第一靶序列和第二靶序列，从而针对SARS-CoV2(COVID-19)和SARS两种病毒进行双重检测。
另一方面，本发明提供了一种多重检测样品中靶核酸的方法，所述方法包 括将样品与核酸检测组合物接触，所述核酸检测组合物包括Cas蛋白、gRNA和单链核酸检测器；所述gRNA包括与所述Cas蛋白结合的区域和与靶核酸上的靶序列杂交的导向序列；检测由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；所述核酸检测组合物选自上述的第一核酸检测组合物、第二核酸检测组合物、第三核酸检测组合物和第四核酸检测组合物中的任意一种、任意两种、任意三种或四种。
另一方面，本发明提供了一种核酸检测组合物，所述核酸检测组合物选自上述的第一核酸检测组合物、第二核酸检测组合物、第三核酸检测组合物和第四核酸检测组合物中的任意一种、任意两种、任意三种或四种。
另一方面，本发明还提供了一种用于检测样品中靶核酸的系统，所述系统包括核酸检测组合物，所述核酸检测组合物包括Cas蛋白、gRNA和单链核酸检测器；所述gRNA包括与所述Cas蛋白结合的区域和与靶核酸上的靶序列杂交的导向序列；所述核酸检测组合物选自上述的第一核酸检测组合物、第二核酸检测组合物、第三核酸检测组合物和第四核酸检测组合物中的任意一种、任意两种、任意三种或四种。
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，所述试剂盒包括核酸检测组合物，所述核酸检测组合物包括Cas蛋白、gRNA和单链核酸检测器；所述gRNA包括与所述Cas蛋白结合的区域和与靶核酸上的靶序列杂交的导向序列。所述核酸检测组合物选自上述的第一核酸检测组合物、第二核酸检测组合物、第三核酸检测组合物和第四核酸检测组合物中的任意一种、任意两种、任意三种或四种。
另一方面，本发明还提供了上述系统或试剂盒在检测样品中靶核酸的应用。如上所述，本发明的系统或试剂盒在对样品中靶核酸进行检测时，可以采用第一核酸检测组合物、第二核酸检测组合物、第三核酸检测组合物和第四核酸检测组合物的一种或任意几种对同一靶序列进行检测，或者针对不同的靶序列进行检测，从而可以实现双重、三重或四重的检测效果。
另一方面，本发明还提供了核酸检测组合物在检测样品中靶核酸的应用，或在制备检测样品中靶核酸的系统或试剂盒中的用途。所述核酸检测组合物选自上述的第一核酸检测组合物、第二核酸检测组合物、第三核酸检测组合物和第四核酸检测组合物中的任意一种、任意两种、任意三种或四种。
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、双链核酸，例如单链DNA、双链DNA、单链RNA、双链RNA。
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。
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，本发明的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。
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。
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。
本发明中，所述gRNA与靶核酸上的靶序列至少有50％的匹配度，优选至少60％，优选至少70％，优选至少80％，优选至少90％。
在一个实施方式中，当所述的靶序列含有一个或多个特征位点(如特定的突变位点或SNP)时，所述的特征位点与gRNA完全匹配。
在一个实施方式中，所述检测方法中可以包含一种或多种导向序列互不相同的gRNA，其靶向不同的靶序列。
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。
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％。
一般定义：
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“寡核苷酸”是指含有3-100个核苷酸的序列，优选，具有3-30个核苷酸，优选，4-20个核苷酸，更优选，5-15个核苷酸。
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。
核酸类似物
如本文所用，“核酸类似物”包括但不限于：2’氧甲基(-OCH 3)RNA、锁核酸、桥核酸、吗啉核酸、乙二醇核酸、己糖醇核酸、苏糖核酸、阿拉伯糖核酸、2’甲氧基乙酰基RNA、2’-氟(-F)RNA、2’-氨基RNA、4’-硫RNA及其组合，包括任选的核糖核苷酸或脱氧核糖核苷酸残基。
锁核酸(LNA)：LNA是2’修饰的核苷，其包含连接所述核苷的核糖糖环的C2’和C4’的双基，所述双基限制或锁定核糖环的构象，其结构式如下所示，LNA的碱基可以选自腺嘌呤，胞嘧啶，鸟嘌呤，5-甲基-胞嘧啶，胸腺嘧啶和尿嘧啶。
Figure PCTCN2021114374-appb-000003
2’氧甲基RNA(2’O-Methyl RNA，2’O-methyl，2’-O-甲基取代RNA，-OCH 3)：2’氧甲基RNA是2’修饰的核苷，其在所述核苷的核糖糖环的C2’位连接一个甲氧基(-OCH 3)。2’氧甲基RNA单体结构如下所示，2’氧甲基RNA的碱基可以选自腺嘌呤，胞嘧啶，鸟嘌呤，5-甲基-胞嘧啶，胸腺嘧啶和尿嘧啶。
Figure PCTCN2021114374-appb-000004
2’-氟基修饰的核酸类似物，或者称之为2’-氟RNA，是2’修饰的核苷，其在所述核苷的核糖糖环的C2’位连接一个F(-F)。2’-氟RNA单体结构如下所示，2’-氟RNA的碱基可以选自腺嘌呤，胞嘧啶，鸟嘌呤，5-甲基-胞嘧啶，胸腺嘧啶和尿嘧啶。
Figure PCTCN2021114374-appb-000005
无碱基间隔物
如本文所用，“无碱基间隔物(Spacer)”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，Spacer9，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，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 PCTCN2021114374-appb-000006
所述dSpacer在于核苷酸连接时，可以形成如下的结构：
Figure PCTCN2021114374-appb-000007
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。
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、Cas1 2b、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。
单链核酸检测器
本发明的单链核酸检测器的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特征序列；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特征序列。
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。
附图说明
图1.当单链核酸检测器序列为5’6-FAM//T//T//3’BHQ1时，Cas12i能特异性的切割单链核酸检测器，较其他蛋白表现出更好的可检测信号。
图2.当单链核酸检测器结构为核酸类似物-锁核酸，序列为5’6-FAM//LNA_T//LNA_T//LNA_T//LNA_T//LNA_T//3’BHQ1时，Cas12b能特异性的切割单链核酸检测器，较其他蛋白表现出更好的可检测信号。
图3.当单链核酸检测器序列为5’6-FAM/S//S//C//S//S//3’BHQ1时，Cas12a能特异性的切割单链核酸检测器，较其他蛋白表现出更好的可检测信号。
图4.当单链核酸检测器序列为5’6-FAM/S//S//A//S//S//3’BHQ1时，Cas12a和Cas12b能特异性的切割单链核酸检测器，较其他蛋白表现出更好的可检测信号，Cas12a的可检测信号强于Cas12b。
图5.当单链核酸检测器序列为5’6-FAM/S//S//T//S//S//3’BHQ1时，Cas12a和Cas12j能特异性的切割单链核酸检测器，较其他蛋白表现出更好的可检测信号。
图6.当单链核酸检测器序列为5’6-FAM/S//S//G//S//S//3’BHQ1时，Cas12a和Cas12b能特异性的切割单链核酸检测器，较其他蛋白表现出更好的可检测信号，Cas12b的可检测信号强于Cas12a。
图7.当单链核酸检测器为核酸类似物-2’氧甲基RNA时，Cas12j能特异性的切割单链核酸检测器，较其他蛋白表现出更好的可检测信号。
图8.利用Cas12i、Cas12j对COVID-19病毒的N基因和S基因进行双重检测：cas12i靶向S基因，reporter为FAM-CT-BHQ1，cas12j靶向N基因，reporter为Cy3-SSTSS-BHQ2；样品中同时存在S基因和N基因时，能检测到两种荧光信号；样品中存在N基因而不存在S基因时，只能检测到对应于Cas12i的FAM荧光信号；样品中存在S基因而不存在N基因时，只能检测到对应于 Cas12j的Cy3荧光信号；样品中不存在S基因或N基因是，两种信号都不能检测到。
图9.利用Cas12a、Cas12b、Cas12i对不同靶核酸进行三重检测。
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；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Cas12i、Cas12j、Cas12a和Cas12b进行核酸检测
本实施方式中，设计不同单链核酸检测器，利用Cas12i、Cas12j、Cas12a和Cas12b进行检测。不同的单链核酸检测器分别为，单链核酸检测器-TT，单链核酸检测器-TT-F，单链核酸检测器-LNA，单链核酸检测器-SSCSS，单链核酸检测器-SSASS，单链核酸检测器-SSTSS，单链核酸检测器-SSGSS和单链核酸检测器-OCH 3。
其中单链核酸检测器-TT的结构为5’6-FAM//T//T//3’BHQ1，单链核酸检测器-TT-F的结构为5’6-FAM//T-F//T-F//3’BHQ1(其中，T-F为对T进行2’氟基修饰的T)，单链核酸检测器-LNA的结构为5’6-FAM//LNA_T//LNA_T//LNA_T//LNA_T//LNA_T//3’BHQ1，单链核酸检测器-SSCSS的结构为5’6-FAM//S//S//C//S//S//3’BHQ1，其中，S为dSpacer，单链核酸检测器-SSASS的结构为5’6-FAM//S//S//A//S//S//3’BHQ1，S为dSpacer，单链核酸检测器-SSTSS的结构为5’6-FAM//S//S//T//S//S//3’BHQ1，S为dSpacer，单链核酸检测器-SSGSS的结构为5’6-FAM//S//S//G//S//S//3’BHQ1，S为dSpacer，单链核酸检测器-OCH 3的结构为5’6-FAM//T-OCH 3//T-OCH 3//T-OCH 3//T-OCH 3//T-OCH 3//3’BHQ1其中，T-OCH 3为2’氧甲基修饰的T。
申请人对Cas12a(SEQ ID No.1)、Cas12b(SEQ ID No.2)、Cas12i(SEQ ID No.3)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 PCTCN2021114374-appb-000008
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Cas12i3-TGW6-g2的序列如SEQ ID No.9所示；
上述Cas12j19-TGW6-g3的序列如SEQ ID No.10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×
100mM DTT 2ul 10mM
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
H2O Up to 20ul  
分别如图1－7所示。
当探针序列为TT时，Cas12i能特异性的切割单链核酸检测器，较其他蛋白表现出更好的可检测信号。
当探针序列为5’6-FAM//T-F//T-F//3’BHQ1时，Cas12i能特异性的切割单链核酸检测器，较其他蛋白表现出更好的可检测信号。
另外，当探针序列为CT(5’6-FAM//C//T//3’BHQ1)时，相较于其他的Cas蛋白，Cas12i同样能够特异性的切割单链核酸检测器，表现出更好的可检测信号。
当探针为核酸类似物-锁核酸(LNA)时，Cas12b能特异性的切割单链核酸 检测器，较其他蛋白表现出更好的可检测信号。
当探针序列为5’6-FAM/S//S//C//S//S//3’BHQ1时，Cas12a能特异性的切割单链核酸检测器，较其他蛋白表现出更好的可检测信号。
当探针序列为5’6-FAM/S//S//A//S//S//3’BHQ1时，Cas12a和Cas12b能特异性的切割单链核酸检测器，较其他蛋白表现出更好的可检测信号。
当探针序列为5’6-FAM/S//S//T//S//S//3’BHQ1时，Cas12a和Cas12j能特异性的切割单链核酸检测器，较其他蛋白表现出更好的可检测信号。
当探针序列为5’6-FAM/S//S//G//S//S//3’BHQ1时，Cas12a和Cas12b能特异性的切割单链核酸检测器，较其他蛋白表现出更好的可检测信号，Cas12b的可检测信号强于Cas12a。
当探针为核酸类似物-2’氧甲基RNA时，Cas12j能特异性的切割单链核酸检测器，较其他蛋白表现出更好的可检测信号。
实施例2、利用Cas12i、Cas12j对COVID-19病毒进行双重检测
利用Cas12i、Cas12j对COVID-19病毒的N基因和S基因进行双重检测：cas12i靶向S基因，reporter为5’6-FAM//C//T//3’BHQ1，gRNA序列为AGAGAAUGUGUGCAUAGUCACACUCAGGAUGUUAACUGCACAG，同SEQ ID No.11所示；cas12j靶向N基因，reporter为5’Cy3//S//S//T//S//S//3’BHQ2，gRNA序列为GUGCUGCUGUCUCCCAGACGGGAGGCAGAACUGCACCGCGACAUUCCGAAGAACGC，同SEQ ID No.12所示。
如图8所示。结果显示，样品中同时存在S基因和N基因时，能检测到两种荧光信号；样品中存在N基因而不存在S基因时，只能检测到对应于Cas12i的FAM荧光信号；样品中存在S基因而不存在N基因时，只能检测到对应于Cas12j的Cy3荧光信号；样品中不存在S基因或N基因时，两种信号都不能检测到。
实施例3、利用Cas12a、Cas12b、Cas12i对不同靶核酸进行三重检测
利用Cas12a、Cas12b、Cas12i对不同靶核酸进行三重检测：
Figure PCTCN2021114374-appb-000009
cas12a靶向EV71 VP1靶核酸，其序列为GTGCACGCAACAAAAGTGAACTCTGCATCAAAGCGCATGT，单链核酸检测器为5’6-FAM//A//dS//dS//T//3’BHQ1(其中 dSpacer为无碱基间隔物)，gRNA为LbCas12a-g71-1，其序列为UAAUUUCUACUAAGUGUAGAU AUGCAGAGUUCACUUUUGUUGCG(加粗是gRNA与蛋白结合的位置，下划线部分是与靶核酸序列匹配的位置)；
cas12b靶向OsTGW6靶核酸，其序列为GATCGTTGGTAGTTCATGCTGCTGTCGGTGAAATAAACATCTCCGGTAAC，单链核酸检测器为5’TAMRA//LNA-T//LNA-T//LNA-T//LNA-T//LNA-T//3’BHQ2(其中LNA-T是指碱基为T的锁核酸)，tracrRNA序列为GUCUAAAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGCCCGUUGAACUUCUCAAAAAGAACGCUCGCUCAGUGUUCUGAC，crRNA序列为GUCGGAUCACUGAGCGAGCGAUCUGAGAAGUGGCAC uuucaccgacagcagcauga(下划线部分是与靶核酸序列匹配的位置)；
cas12i靶向COVID-19 orf1ab靶核酸，其序列为Ggcaccaaattccaaaggtttaccttggtaatcatcttcagtaccatactcatattgag，单链核酸检测器为5’HEX//C//T//3’BHQ1，gRNA为CV19-Lamb-i3g5g，其序列为AGAGAAUGUGUGCAUAGUCACAC ccaaggUaaaccUUUggaaUUUgg(加粗是gRNA与蛋白结合的位置，下划线部分是与靶核酸序列匹配的位置)。
如图9中，图左侧是指在该体系中加入的靶核酸，以对应酶的缩写表示，如“ABI”是指体系中加入Cas12a(A)蛋白所检测的EV71 VP1靶核酸、Cas12b(B)蛋白所检测的OsTGW6靶核酸和Cas12i(I)蛋白所检测的COVID-19 orf1ab靶核酸；图上侧是指该体系中的Cas蛋白识别到靶核酸后激活旁路切割活性、特异切割单链核酸检测器所产生的荧光信号，如“Cas12-FAM”是指，此体系中的Cas12蛋白识别到EV71 VP1靶核酸后旁路切割活性被激活，特异性的切割单链核酸检测器5’6-FAM//A//S//S//T//3’BHQ1，产生的FAM荧光强度，颜色越深则信号越强。
具体的例如，第一行，当向体系中加入Cas12a(A)蛋白所检测的EV71 VP1靶核酸、Cas12b(B)蛋白所检测的OsTGW6靶核酸和Cas12i(I)蛋白所检测的COVID-19 orf1ab靶核酸时，可以检测到Cas12a对应的FAM荧光、Cas12b对应的TAMRA荧光和Cas12i对应的HEX荧光。
检测结果证明Cas12a、Cas12b和Cas12i对于单链核酸检测器的偏好性各异，可以用于三重核酸的检测。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

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核酸检测组合物接触，所述核酸检测组合物包括Cas蛋白、gRNA和单链核酸检测器；所述gRNA包括与所述Cas蛋白结合的区域和与靶核酸上的靶序列杂交的导向序列；检测由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；
    所述核酸检测组合物选自第一核酸检测组合物、第二核酸检测组合物、第三核酸检测组合物和第四核酸检测组合物中的任意一种、任意两种、任意三种或四种；
    所述第一核酸检测组合物包括Cas12i，可以结合Cas12i以及与靶核酸上的第一靶序列杂交的第一gRNA，和第一单链核酸检测器；
    所述第二核酸检测组合物包括Cas12b，可以结合Cas12b以及与靶核酸上的第二靶序列杂交的第二gRNA，和第二单链核酸检测器；
    所述第三核酸检测组合物包括Cas12a，可以结合Cas12a以及与靶核酸上的第三靶序列杂交的第三gRNA，和第三单链核酸检测器；
    所述第四核酸检测组合物包括Cas12j，可以结合Cas12j以及与靶核酸上的第四靶序列杂交的第四gRNA，和第四单链核酸检测器；
    所述第一单链核酸检测器含有至少两个连续的核苷酸，所述核苷酸为核糖核苷酸、脱氧核糖核苷酸、核酸类似物中的一种或几种；
    所述第二单链核酸检测器选自含有无碱基间隔物的单链核酸检测器，所述含有无碱基间隔物的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；或者，所述第二单链核酸检测器的核酸结构为核酸类似物，所述核酸类似物为锁核酸(LNA)；
    所述第三单链核酸检测器是含有无碱基间隔物的单链核酸检测器；所述含有无碱基间隔物的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；
    所述第四单链核酸检测器选自含有无碱基间隔物的单链核酸检测器，所述含有无碱基间隔物的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；或者，所述第四单链核酸检测器的核酸结构为核酸类似物，所述核酸类似物为2’氧甲基RNA。
  2. 一种对样品中的靶核酸进行多重检测的方法，所述方法包括将样品与核酸检测组合物接触，所述核酸检测组合物包括Cas蛋白、gRNA和单链核酸检测器；所述gRNA包括与所述Cas蛋白结合的区域和与靶核酸上的靶序列杂交的导向序列；检测由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；
    所述核酸检测组合物选自权利要求1中所述的第一核酸检测组合物、第二核酸检测组合物、第三核酸检测组合物和第四核酸检测组合物中的任意两种、任意三种或四种。
  3. 根据权利要求1或2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，胶体相变/分散，电化学检测和基于半导体的检测。
  4. 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。
  5. 根据权利要求1-4任一所述的方法，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；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。
  6. 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，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。
  7. 根据权利要求1-6任一所述的方法，其特征在于，所述方法还包括从样品中获得核酸的步骤。
  8. 一种用于检测样品中靶核酸的系统或组合物或试剂盒，所述系统或组合物或试剂盒包括核酸检测组合物，所述核酸检测组合物包括Cas蛋白、gRNA和单链核酸检测器；所述gRNA包括与所述Cas蛋白结合的区域和与靶核酸上的靶序列杂交的导向序列；所述核酸检测组合物选自权利要求1中所述的第一核酸检测组合物、第二核酸检测组合物、第三核酸检测组合物和第四核酸检测组合物中的任意一种、任意两种、任意三种或四种。
  9. 一种用于对样品中靶核酸进行多重检测的系统或组合物或试剂盒，所述系统或组合物或试剂盒包括核酸检测组合物，所述核酸检测组合物包括Cas蛋白、gRNA和单链核酸检测器；所述gRNA包括与所述Cas蛋白结合的区域和与靶核酸上的靶序列杂交的导向序列；所述核酸检测组合物选自权利要求1中所述的第一核酸检测组合物、第二核酸检测组合物、第三核酸检测组合物和第四核酸检测组合物中的任意两种、任意三种或四种。
  10. 权利要求8-9任一所述的系统或组合物或试剂盒在检测样品中靶核酸的应用。
PCT/CN2021/114374 2020-08-28 2021-08-24 基于crispr技术进行多重核酸检测的方法 WO2022042568A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/648,180 US20220136075A1 (en) 2020-08-28 2022-01-18 Method for multiplex nucleic acid detection based on clustered regularly interspaced short palindromic repeat

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202010888036 2020-08-28
CN202010888036.3 2020-08-28
CN202110236947.2A CN112795625B (zh) 2020-08-28 2021-03-03 基于crispr技术进行多重核酸检测的方法
CN202110236947.2 2021-03-03

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/648,180 Continuation US20220136075A1 (en) 2020-08-28 2022-01-18 Method for multiplex nucleic acid detection based on clustered regularly interspaced short palindromic repeat

Publications (1)

Publication Number Publication Date
WO2022042568A1 true WO2022042568A1 (zh) 2022-03-03

Family

ID=75816439

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/114374 WO2022042568A1 (zh) 2020-08-28 2021-08-24 基于crispr技术进行多重核酸检测的方法

Country Status (3)

Country Link
US (1) US20220136075A1 (zh)
CN (4) CN113789365B (zh)
WO (1) WO2022042568A1 (zh)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113373264B (zh) * 2021-06-10 2022-04-12 安徽医科大学 一种基于CRISPR/Cas系统的新型冠状病毒双靶标快速检测方法及试剂盒
CN113502351B (zh) * 2021-06-29 2023-11-21 中国人民解放军疾病预防控制中心 一种crRNA及用于CRISPR-Cas12a检测人腺病毒核酸的方法
CN115044649A (zh) * 2021-08-11 2022-09-13 山东舜丰生物科技有限公司 一种改进的基于crispr技术检测靶核酸的方法
CN114634972B (zh) * 2022-05-19 2022-08-26 舜丰生物科技(海南)有限公司 利用Cas酶进行核酸检测的方法
WO2023231456A1 (zh) * 2022-05-31 2023-12-07 山东舜丰生物科技有限公司 一种优化的Cas蛋白及其应用
WO2024040874A1 (zh) * 2022-08-22 2024-02-29 山东舜丰生物科技有限公司 突变的Cas12j蛋白及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019011022A1 (zh) * 2017-07-14 2019-01-17 上海吐露港生物科技有限公司 一种Cas蛋白的用途及靶标核酸分子的检测方法和试剂盒
WO2019104058A1 (en) * 2017-11-22 2019-05-31 The Regents Of The University Of California Type v crispr/cas effector proteins for cleaving ssdnas and detecting target dnas
CN110846386A (zh) * 2019-11-15 2020-02-28 浙江大学 核酸的多重特异性可视化检测方法及装置
WO2020051562A2 (en) * 2018-09-07 2020-03-12 Beam Therapeutics Inc. Compositions and methods for improving base editing

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030165888A1 (en) * 2001-07-18 2003-09-04 Brown Bob D. Oligonucleotide probes and primers comprising universal bases for diagnostic purposes
EP3875469A4 (en) * 2018-10-29 2022-08-17 China Agricultural University NEW CRISPR/CAS12F ENZYME AND SYSTEM

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019011022A1 (zh) * 2017-07-14 2019-01-17 上海吐露港生物科技有限公司 一种Cas蛋白的用途及靶标核酸分子的检测方法和试剂盒
WO2019104058A1 (en) * 2017-11-22 2019-05-31 The Regents Of The University Of California Type v crispr/cas effector proteins for cleaving ssdnas and detecting target dnas
WO2020051562A2 (en) * 2018-09-07 2020-03-12 Beam Therapeutics Inc. Compositions and methods for improving base editing
CN110846386A (zh) * 2019-11-15 2020-02-28 浙江大学 核酸的多重特异性可视化检测方法及装置

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PAUSCH PATRICK, AL-SHAYEB BASEM, BISOM-RAPP EZRA, TSUCHIDA CONNOR A., LI ZHENG, CRESS BRADY F., KNOTT GAVIN J., JACOBSEN STEVEN E.: "CRISPR-CasΦ from huge phages is a hypercompact genome editor", SCIENCE, vol. 369, no. 6501, 17 July 2020 (2020-07-17), US , pages 333 - 337, XP055862891, ISSN: 0036-8075, DOI: 10.1126/science.abb1400 *

Also Published As

Publication number Publication date
CN113801918A (zh) 2021-12-17
CN113789365B (zh) 2022-11-22
CN113801917A (zh) 2021-12-17
CN113789365A (zh) 2021-12-14
CN112795625A (zh) 2021-05-14
CN112795625B (zh) 2022-02-08
CN113801917B (zh) 2022-11-22
US20220136075A1 (en) 2022-05-05
CN113801918B (zh) 2022-11-29

Similar Documents

Publication Publication Date Title
WO2022042568A1 (zh) 基于crispr技术进行多重核酸检测的方法
CN111996236B (zh) 基于crispr技术进行靶核酸检测的方法
CN111690720B (zh) 利用修饰的单链核酸进行靶核酸检测的方法
CN111690773B (zh) 利用新型Cas酶进行目标核酸检测的方法和系统
EP3653722A1 (en) Application of cas protein, method for detecting target nucleic acid molecule and kit
JP2018064585A (ja) 分子計数のための組成物およびキット
WO2022012423A1 (zh) 利用含有无碱基间隔物的核酸检测器检测靶核酸的方法
US9845495B2 (en) Method and kit for detecting target nucleic acid
CN111690717A (zh) 基于crispr技术进行目标核酸检测的方法和系统
EP3719142A2 (en) Method for amplifying target nucleic acid and composition for amplifying target nucleic acid
CN111733216A (zh) 一种提高目标核酸检测效率的方法
CN113667718B (zh) 利用双链核酸检测器进行靶核酸检测的方法
CN111876469B (zh) 利用核酸类似物进行靶核酸检测的方法
CN113913499A (zh) 利用Cas12j效应蛋白检测目标突变的方法
CN113913498A (zh) 一种基于crispr技术检测目标突变的方法
US20230031670A1 (en) Method and kit for detection of polynucleotide
WO2021254267A1 (zh) 利用核酸类似物或碱基修饰物进行靶核酸检测的方法
CN114634972B (zh) 利用Cas酶进行核酸检测的方法
CN113234795B (zh) 利用Cas蛋白进行核酸检测的方法
CN113293198B (zh) 基于crispr技术进行靶核酸多重检测的方法
CN114517224A (zh) 一种利用优化的单链核酸检测器进行核酸检测的方法
CN112608913B (zh) 一种基于C2c2的基因表达调控系统及其应用
CN113913497A (zh) 利用碱基修饰的单链核酸进行靶核酸检测的方法
CN115044649A (zh) 一种改进的基于crispr技术检测靶核酸的方法
EP4265741A1 (en) Multiplexable crispr-cas9-based virus detection method

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21860403

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21860403

Country of ref document: EP

Kind code of ref document: A1