WO2021254284A1 - Procédé et système pour effectuer une détection d'acide nucléique cible à l'aide d'une nouvelle enzyme cas - Google Patents

Procédé et système pour effectuer une détection d'acide nucléique cible à l'aide d'une nouvelle enzyme cas Download PDF

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WO2021254284A1
WO2021254284A1 PCT/CN2021/099831 CN2021099831W WO2021254284A1 WO 2021254284 A1 WO2021254284 A1 WO 2021254284A1 CN 2021099831 W CN2021099831 W CN 2021099831W WO 2021254284 A1 WO2021254284 A1 WO 2021254284A1
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nucleic acid
exonuclease
detected
target nucleic
detection
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PCT/CN2021/099831
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Chinese (zh)
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梁亚峰
段志强
孙洁
刘锐恒
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山东舜丰生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • 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

Definitions

  • the present invention relates to the field of nucleic acid detection, in particular to a method and system for target nucleic acid detection using a novel Cas enzyme.
  • Nucleic acid detection methods have important applications, such as pathogen detection and genetic disease detection.
  • pathogen detection since each pathogen microorganism has its unique characteristic nucleic acid molecular sequence, nucleic acid molecular detection for specific species can be developed, also known as nucleic acid diagnostics (NADs, nucleic acid diagnostics). Microbial contamination detection, human pathogenic bacteria infection and other fields are of great significance.
  • 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 functions at the genome level provides a new perspective for modern molecular biology. Among them, SNPs are closely related to biological functions, evolution, and diseases. Therefore, the development of SNPs detection and analysis techniques Particularly important.
  • the current 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 methods, Southern, Northern, dot blot, fluorescent PCR detection technology, LAMP loop-mediated isothermal amplification technology, recombinase polymerase amplification technology (RPA) and other methods.
  • RPA recombinase polymerase amplification technology
  • CRISPR gene editing technology emerged.
  • Zhang Feng's team developed a new nucleic acid diagnostic technology (SHERLOCK technology) with Cas13 as the core based on RPA technology.
  • the present invention provides a method, system and kit for detecting target nucleic acid using a novel Cas enzyme.
  • the present invention provides a method for detecting whether there is a characteristic sequence to be detected in a target nucleic acid using a novel Cas enzyme, the method comprising:
  • the exonuclease is used to digest the target nucleic acid to form a nucleic acid sequence containing at least part of a single-stranded nucleic acid;
  • the gRNA can target the characteristic sequence to be detected, the Cas protein recognizes the characteristic sequence to be detected under the action of the gRNA, and the Cas protein recognizes the characteristic sequence to be detected and then stimulates the pair Cleavage activity of single-stranded nucleic acid detector;
  • the Cas protein cleaves the single-stranded nucleic acid detector, and the single-stranded nucleic acid detector is cleaved by the Cas protein and shows a detectable difference compared to before the single-stranded nucleic acid detector is cleaved by the Cas protein;
  • step (4) Test whether the detectable difference described in step (4) can be detected; if the detectable difference described in step (4) can be detected, it reflects that the target nucleic acid contains the characteristic sequence to be detected; or, If the detectable difference described in step (4) cannot be detected, it reflects that the target nucleic acid does not contain the characteristic sequence to be detected.
  • the exonuclease is used to digest the target nucleic acid to form a single-stranded nucleic acid. As shown in Figure 1, due to the action of the exonuclease, the double-stranded target nucleic acid will be cleaved into a partially single-stranded nucleic acid. The form of nucleic acid.
  • the target nucleic acid includes DNA and RNA, preferably a double-stranded nucleic acid.
  • the target nucleic acid is derived from samples such as viruses, bacteria, microorganisms, soil, water sources, human bodies, animals, and plants.
  • the target nucleic acid is a product amplified by PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM.
  • the method further includes the step of obtaining the target nucleic acid from the sample.
  • the characteristic sequence to be detected is a virus-specific sequence, a bacterial-specific sequence, a disease-related characteristic sequence, a specific mutation site or a SNP site; preferably, the virus is a plant virus Or animal virus; preferably, the virus is a coronavirus, preferably SARS, SARS-CoV2 (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-Cov.
  • the sample from which the target nucleic acid is derived is a certain virus, bacteria, or is infected with a certain virus, bacteria, or disease, or has a specific mutation site or SNP Site.
  • the target nucleic acid is derived from a cell, for example, from a cell lysate.
  • the Cas protein is a protein with double-stranded and/or single-stranded nucleic acid or nucleic acid analog cleavage activity.
  • the Cas protein is selected from type V CRISPR/CAS effector proteins, including: Cas12, Cas14 family proteins or mutants thereof.
  • the Cas protein mutant includes amino acid substitutions, deletions or substitutions, and the mutant at least retains its trans-cleavage activity.
  • the mutant has Cis and trans cleavage activity.
  • the Cas protein is preferably the Cas12 family, including but not limited to Cas12a, Cas12b, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j; preferably one of Cas12a, Cas12b, Cas12i, Cas12j or Any number.
  • the single-stranded nucleic acid detector is selected from one or more of single-stranded DNA, single-stranded RNA, or single-stranded DNA-RNA hybrids; more preferably, the single-stranded nucleic acid detector also includes nucleic acid Modifications or nucleic acid analogs.
  • the Cas protein is Cas12i and/or Cas12j
  • the single-stranded nucleic acid detector is selected from one or more of single-stranded DNA, single-stranded RNA, or single-stranded DNA-RNA hybrids;
  • the single-stranded nucleic acid detector further includes nucleic acid modifications or nucleic acid analogs.
  • the Cas protein is Cas12a and/or Cas12b
  • the single-stranded nucleic acid detector is selected from one or more of single-stranded DNA, single-stranded RNA, or single-stranded DNA-RNA hybrids;
  • the single-stranded nucleic acid detector further includes nucleic acid modifications or nucleic acid analogs.
  • the Cas protein is a Cas14 family protein, preferably Cas14a and/or Cas14b;
  • the single-stranded nucleic acid detector is selected from one of single-stranded DNA, single-stranded RNA, or single-stranded DNA-RNA hybrids.
  • the single-stranded nucleic acid detector also includes nucleic acid modifications or nucleic acid analogs.
  • Cas12i is selected from proteins consisting of the following sequences:
  • the Cas12i protein further includes 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 trans-active protein.
  • the Cas12j is preferably a protein composed of the following sequence:
  • the Cas12j protein further includes 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 trans-active protein.
  • the Cas12a is preferably a protein composed of the following sequence:
  • the Cas12a protein further includes 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 trans-active protein.
  • the Cas12b is preferably a protein composed of the following sequence:
  • the Cas12b protein further includes 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 trans-active protein.
  • the gRNA includes a sequence that targets the characteristic sequence to be detected (a targeting sequence) and a sequence that recognizes the Cas protein (a direct repeat sequence or a part thereof).
  • the targeting sequence includes 10-40 bp; preferably, 12-30 bp; preferably, 15-25 bp; preferably, 16-18 bp.
  • the gRNA has a matching degree of at least 50% with the characteristic sequence to be detected, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.
  • the characteristic sequence contains one or more characteristic sites (such as specific mutation sites or SNPs)
  • the characteristic sites are completely matched with the gRNA.
  • the detection method may include one or more gRNAs with different targeting sequences, which target different characteristic sequences.
  • the exonuclease is selected from 5'exonuclease or 3'exonuclease; preferably, the exonuclease is 5'exonuclease; or It is 5' ⁇ 3' exonuclease or 3' ⁇ 5' exonuclease, preferably, 5' ⁇ 3' exonuclease.
  • the exonuclease is selected from: T5 exonuclease, T7 exonuclease, lambda exonuclease or exonuclease VIII.
  • the identifying the characteristic sequence to be detected includes combining and/or cutting the characteristic sequence to be detected.
  • the detectable distinction is realized by the following methods: visual-based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization, colloidal phase change/dispersion, electrochemical detection, and semiconductor-based detection Detection.
  • the method of the present invention further includes the step of measuring the detectable difference 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 cutting the single-stranded nucleic acid detector to produce a detectable difference.
  • the detectable difference can be any signal generated when the single-stranded nucleic acid detector is cleaved. For example, detection based on gold nanoparticles, fluorescence polarization, colloidal phase transition/dispersion, electrochemical detection, semiconductor-based sensing.
  • the detectable difference can be read out in any suitable way, including but not limited to: measurement of detectable fluorescent signal, gel electrophoresis detection (by detecting the change of bands on the gel), vision or sensor-based Detection of the presence or absence of color, or the difference in color (for example, based on gold nanoparticles) and the difference in electrical signals.
  • the detection of the detectable difference is achieved in the following manner: the 5'end and 3'end of the single-stranded nucleic acid detector are respectively provided with different reporter groups, when the single-stranded nucleic acid detector After being cut, it can show a detectable report signal, and the presence or absence of the report signal reflects whether the target nucleic acid contains the characteristic sequence to be detected; if the report signal can be detected, it reflects that the target nucleic acid contains the characteristic sequence to be detected; or , If the report signal cannot be detected, it reflects that the target nucleic acid does not contain the characteristic sequence to be detected.
  • a fluorescent group and a quenching group are provided at both ends of a single-stranded nucleic acid detector.
  • the single-stranded nucleic acid detector When the single-stranded nucleic acid detector is cut, it can show a detectable fluorescent signal, and the presence or absence of the fluorescent signal reflects the target Whether the nucleic acid contains the characteristic sequence to be detected; if the fluorescent signal can be detected, it reflects that the target nucleic acid contains the characteristic sequence to be detected; or, if the fluorescent signal cannot be detected, it reflects that the target nucleic acid does not contain the characteristic sequence to be detected.
  • the fluorescent group is selected from one or more of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460; the quenching group is selected From one or more of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.
  • the detection of the detectable difference can also be achieved in other ways: the 5'end and the 3'end of the single-stranded nucleic acid detector are respectively provided with different labeling molecules, and the detection is carried out by colloidal gold. , Detecting the colloidal gold test results before the single-stranded nucleic acid detector is cleaved by the Cas protein and after being cleaved by the Cas protein to reflect whether the target nucleic acid contains the characteristic sequence to be detected; before the single-stranded nucleic acid detector is cleaved by the Cas protein It will show different color development results on the detection line and quality control line of colloidal gold after being cut by Cas protein.
  • the molar ratio of the Cas protein to gRNA is (0.8-1.2):1.
  • the final concentration of the Cas protein is 20-200 nM, preferably 30-100 nM, more preferably 40-80 nM, more preferably 50 nM.
  • the final concentration of the amount of gRNA is 20-200 nM, preferably, 30-100 nM, more preferably, 40-80 nM, more preferably, 50 nM.
  • the final concentration of the amount of the target nucleic acid is 5-100 nM, preferably, 10-50 nM.
  • the dosage of the single-stranded nucleic acid detector has a final concentration of 100-1000 nM, preferably, 150-800 nM, preferably, 200-800 nM, preferably, 200-500 nM, preferably, 200-300 nM.
  • the final concentration of the amount of exonuclease is 0.1-3.0 U/ ⁇ l, preferably, 0.2-2.0 U/ ⁇ l, more preferably, 0.5-1.0 U/ ⁇ l.
  • the single-stranded nucleic acid detector includes single-stranded DNA, single-stranded RNA, or single-stranded DNA-RNA hybrid. In other embodiments, the single-stranded nucleic acid detector includes single-stranded DNA, single-stranded RNA, or a mixture of any two or three of single-stranded DNA-RNA hybrids, for example, single-stranded DNA and single-stranded RNA. Composition, single-stranded DNA and single-stranded DNA-RNA hybrid composition, single-stranded RNA and single-stranded DNA-RNA composition.
  • the single-stranded nucleic acid detector also includes nucleic acid modifications or nucleic acid analogs; such as base modification, backbone modification, sugar modification, etc., to provide nucleic acids with new or enhanced features (such as improved stability). sex).
  • nucleic acid modifications or nucleic acid analogs such as base modification, backbone modification, sugar modification, etc.
  • suitable modifications include modified nucleic acid backbones and non-natural nucleoside linkages.
  • Nucleic acids with modified backbones include those that retain phosphorus atoms in the backbone and those that do not have phosphorus atoms in the backbone.
  • Suitable modified oligonucleotide backbones containing phosphorus atoms include phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl groups. Phosphonate.
  • Suitable base modification types include one or more of deamination modification, methylation, acetylation, hydrogenation, fluorination, or sulfurization modification.
  • the single-stranded nucleic acid detector contains one or more phosphorothioate and/or heteroatom nucleoside bonds.
  • Suitable nucleic acid analogs include, but are not limited to: 2'-O-methyl substituted RNA, locked nucleic acid, bridge nucleic acid, morpholine nucleic acid, glycol nucleic acid, hexitol nucleic acid, threose nucleic acid, arabinose nucleic acid, 2' Oxymethyl RNA, 2'methoxyacetyl RNA, 2'-fluoro RNA, 2'-amino RNA, 4'-sulfur RNA, and combinations thereof.
  • the single-stranded nucleic acid detector has 2-300 bases, preferably 3-200 bases, preferably 4-100 bases, and more preferably 5-50 bases.
  • the method can be used for the quantitative detection of the characteristic sequence to be detected.
  • the present invention also provides a system for detecting whether there is a characteristic sequence to be detected in a target nucleic acid based on CRISPR technology.
  • the system includes: an exonuclease, gRNA, Cas protein and a single-stranded nucleic acid detector;
  • the exonuclease is used to digest the target nucleic acid to form a nucleic acid sequence containing at least part of a single-stranded nucleic acid;
  • the gRNA can target the characteristic sequence to be detected, the Cas protein recognizes the characteristic sequence to be detected under the action of the gRNA, and the Cas protein stimulates single-stranded nucleic acid detection after recognizing the characteristic sequence to be detected Cutting activity of the device;
  • the Cas protein cleaves the single-stranded nucleic acid detector to produce a detectable difference
  • 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 characteristic sequence to be detected.
  • the present invention also provides a CRISPR technology-based kit for detecting whether there is a characteristic sequence to be detected in a target nucleic acid, the kit comprising: exonuclease, gRNA, Cas protein and single-stranded Nucleic acid detector.
  • kit also includes primers for amplifying the target nucleic acid.
  • the present invention also provides the use of the above-mentioned system or the above-mentioned kit in diagnosing whether there is a characteristic sequence to be detected in the sample to be tested.
  • the use includes obtaining a target nucleic acid from a sample to be tested, and further detecting whether there is a characteristic sequence to be detected in the target nucleic acid.
  • NASBA nucleic acid sequencing-based amplification
  • RPA recombinase polymerase amplification
  • LAMP loop-mediated isothermal amplification
  • SDA strand displacement amplification
  • HDA helicase-dependent amplification Increase
  • NEAR Nickase Amplification Reaction
  • MDA Multiple Displacement Amplification
  • RCA Rolling Circle Amplification
  • LCR Ligase Chain Reaction
  • RAM Derivative Amplification Method
  • the characteristic sequence to be detected is a virus-specific sequence, a bacterial-specific sequence, a disease-related characteristic sequence, a specific mutation site or a SNP site; preferably, the virus is a plant Virus or animal virus; preferably, the virus is a coronavirus, preferably SARS, SARS-CoV2 (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-Cov.
  • the sample from which the target nucleic acid is derived is a certain virus, bacteria, or is infected with a certain virus, bacteria, or disease, or has a specific mutation site or SNP Site.
  • the present invention also provides a system for detecting whether there is a characteristic sequence to be detected in a target nucleic acid, the system including a detection device, a reaction system and a detection agent;
  • the reaction system includes exonuclease, gRNA, Cas protein and single-stranded nucleic acid detector;
  • the exonuclease is used to digest the target nucleic acid to form a nucleic acid sequence containing at least part of a single-stranded nucleic acid;
  • the gRNA can target the characteristic sequence to be detected, and the Cas protein is under the action of the gRNA Identifying the characteristic sequence to be detected, and cutting the single-stranded nucleic acid detector after the Cas protein recognizes the characteristic sequence to be detected;
  • One end of the single-stranded nucleic acid detector is connected with a first labeling molecule, and the other end is connected with a second labeling molecule;
  • the detection agent is connected with a first binding molecule, and the first labeling molecule can bind to the first binding molecule;
  • the detection device (preferably, a test strip) is provided with a first detection line and a second detection line, the first detection line is provided with a first substance that can capture the first binding molecule, and the second detection line is provided There is a second substance that can capture the second label molecule.
  • the detection device is a test paper, preferably a lateral flow test paper.
  • the first detection line is downstream of the second detection line.
  • the detection agent is preferably placed on the detection device, more preferably, according to the direction of flow, the detection agent is placed upstream of the first detection line and the second detection line.
  • the first detection line and the second detection line also become a quality control line and a detection line.
  • reaction system after the reaction system has reacted for a period of time, at least part or all of the detection agent is contacted with the reaction system.
  • the detection agent is colloidal metal.
  • the colloidal metal material may include water-insoluble metal particles or metal compounds dispersed in liquid, hydrosol or metal sol.
  • Preferred metals include gold, silver, aluminum, ruthenium, zinc, iron, nickel, and calcium.
  • Other suitable metals include the following various oxidation states: lithium, sodium, magnesium, potassium, scandium, titanium, vanadium, chromium, manganese, cobalt, copper, gallium, strontium, niobium, molybdenum, palladium, indium, tin, Tungsten, rhenium, platinum and gadolinium; in a preferred embodiment, the colloidal metal is colloidal gold.
  • the detection agent is a nanometer particle, preferably, 2nm-100nm, more preferably, 10nm-80nm, more preferably, 15nm, 20nm, 30nm.
  • the detection agent After the detection agent contacts the reaction system, according to the reaction result of the reaction system, the detection agent will show different capture signals on the first detection line and the second detection line.
  • the single-stranded nucleic acid detector will not be cleaved. After the detection agent comes into contact with the reaction system, more signals will be captured by the second substance and accumulated at the second detection line. Fewer signals appear on one detection line or no signals appear on the first detection line.
  • the single-stranded nucleic acid detector will be cut. After the detection agent comes into contact with the reaction system, more signals will be captured by the first substance and accumulated at the first detection line. Less signal appears on the detection line or no signal appears on the second detection line.
  • the first labeling molecule is fluorescein, for example, fluorescein isothiocyanate (FITC) or carboxyfluorescein (FAM), or digoxigenin (DIG), 5-carboxytetramethyl Rhodamine (TAMRA).
  • the second labeling molecule is Biotin; preferably, the first labeling molecule is placed at the 5'end of the single-stranded oligonucleotide, and the second labeling molecule is placed on the single-stranded oligonucleotide. The 3'end of the nucleotide.
  • the first binding molecule is a first antibody that can bind to the first labeling molecule
  • the first substance is a second antibody that can bind to the first binding molecule.
  • the first antibody is an anti-FITC antibody, an anti-FAM antibody, an anti-digoxigenin antibody, or an anti-5-carboxytetramethylrhodamine antibody.
  • the second substance is avidin that can bind to biotin, preferably streptavidin.
  • the present invention also provides a kit for detecting whether there is a characteristic sequence to be detected in a target nucleic acid containing the above-mentioned system.
  • kit also includes primers for amplifying the target nucleic acid.
  • the present invention also provides the use of the above-mentioned system or the above-mentioned kit in diagnosing whether there is a characteristic sequence to be detected in the sample to be tested.
  • the use includes obtaining a target nucleic acid from a sample to be tested, and further detecting whether there is a characteristic sequence to be detected in the target nucleic acid.
  • NASBA nucleic acid sequencing-based amplification
  • RPA recombinase polymerase amplification
  • LAMP loop-mediated isothermal amplification
  • SDA strand displacement amplification
  • HDA helicase-dependent amplification Increase
  • NEAR Nickase Amplification Reaction
  • MDA Multiple Displacement Amplification
  • RCA Rolling Circle Amplification
  • LCR Ligase Chain Reaction
  • RAM Derivative Amplification Method
  • the characteristic sequence to be detected is a virus-specific sequence, a bacterial-specific sequence, a disease-related characteristic sequence, a specific mutation site or a SNP site; preferably, the virus is a plant Virus or animal virus; preferably, the virus is a coronavirus, preferably SARS, SARS-CoV2 (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-Cov.
  • the sample from which the target nucleic acid is derived is a certain virus, bacteria, or is infected with a certain virus, bacteria, or disease, or has a specific mutation site or SNP Site.
  • the present invention also provides the use of the above system to detect whether there is a characteristic sequence to be detected in the target nucleic acid.
  • the present invention also provides the use of the above-mentioned system in diagnosing or detecting whether there is a characteristic sequence to be detected in the sample to be tested.
  • the use includes obtaining a target nucleic acid from a sample to be tested, and further detecting whether there is a characteristic sequence to be detected in the target nucleic acid.
  • the molar ratio of the Cas protein to gRNA is (0.8-1.2):1.
  • the final concentration of the Cas protein is 20-200 nM, preferably 30-100 nM, more preferably 40-80 nM, more preferably 50 nM.
  • the final concentration of the amount of gRNA is 20-200 nM, preferably, 30-100 nM, more preferably, 40-80 nM, more preferably, 50 nM.
  • the final concentration of the amount of the target nucleic acid is 5-100 nM, preferably, 10-50 nM.
  • the dosage of the single-stranded nucleic acid detector has a final concentration of 100-1000 nM, preferably, 150-800 nM, preferably, 200-800 nM, preferably, 200-500 nM, preferably, 200-300 nM.
  • the final concentration of the amount of exonuclease is 0.1-3.0 U/ ⁇ l, preferably, 0.2-2.0 U/ ⁇ l, more preferably, 0.5-1.0 U/ ⁇ l.
  • amino acid refers to a carboxylic acid containing an amino group.
  • Various proteins in organisms are composed of 20 basic amino acids.
  • nucleic acid refers to DNA, RNA or a hybrid thereof, which may be double-stranded or single-stranded.
  • each molecule is at that position
  • the identity of the amino acid sequence can be determined by conventional methods, see, for example, Smith and Waterman, 1981, Adv. Appl. Math. 2: 482 Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA 85 :2444, Thompson et al., 1994, Nucleic Acids Res 22:467380, etc., are determined by computerized operating algorithms (GAP, BESTFIT, FASTA, and TFASTA, Genetics Computer Group in the Wisconsin Genetics software package).
  • the BLAST algorithm available from the National Center for Biotechnology Information NCBI www.ncbi.nlm.nih.gov/
  • NCBI www.ncbi.nlm.nih.gov/ can also be used, and the default parameters are used to determine.
  • CRISPR refers to clustered regularly interspaced short palindromic repeats (Clustered regularly interspaced short palindromic repeats), which are 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 (avidin) also known as avidin
  • avidin is a basic glycoprotein that has 4 binding sites with extremely high affinity to biotin.
  • the commonly used avidin is streptavidin. The extremely strong affinity of biotin and avidin can be used to amplify or enhance the detection signal in the detection system.
  • biotin is easy to covalently bond with proteins (such as antibodies, etc.), and the avidin molecule bound to the enzyme reacts with the biotin molecule bound to the specific antibody, which not only plays a multi-stage amplification effect, but also because When the enzyme encounters the corresponding substrate, it will be colored by the catalytic action, so as to achieve the purpose of detecting unknown antigen (or antibody) molecules.
  • the "characteristic sequence” or “characteristic sequence to be detected” or “characteristic sequence to be detected” can be used interchangeably, and both refer to a nucleic acid sequence that characterizes the specificity or certain unique characteristics of an organism. Hybridization with the gRNA guide sequence promotes the formation of CRISPR complexes.
  • the said characteristic sequence is a DNA polynucleotide, and its quantity can include the part complementary to the gRNA guide sequence, or it can be equal to or slightly less than the part complementary to the gRNA guide sequence.
  • the organisms include animals, plants, and microorganisms.
  • the microorganisms include bacteria, fungi, yeasts, protozoa, parasites or viruses.
  • the characteristic sequence may be a nucleic acid sequence that characterizes the characteristics of the virus (if the virus is an RNA sequence, it also includes a DNA sequence formed by reverse transcription); for example, the characteristic sequence may be a sequence containing specific mutation sites For example, gene mutation sites that induce tumors in animal cells, or certain gene mutation sites that change plant traits in plants (such as specific mutation sites that confer herbicide resistance to the ALS protein).
  • the virus includes a double-stranded RNA virus, a positive sense RNA virus, a negative sense RNA virus, a retrovirus, or a combination thereof, or the virus infection is caused by a Coronaviridae virus, a picornavirus ( Picornaviridae virus, Caliciviridae virus, Flaviviridae virus, Togaviridae virus, Filoviridae, Paramyxoviridae, Pneumoviridae ( Pneumoviridae, Rhabdoviridae, Arenaviridae, Bunyaviridae, Orthomyxoviridae or Delta virus, or the virus infection is caused by Coronavirus , SARS, Poliovirus, Rhinovirus, Hepatitis A, Norwalkvirus, Yellow fever virus, West Nile virus ), Hepatitis C virus, Dengue fever virus, Zika virus, Rubella virus, Ross River virus, Sindbis virus ), Chikungunya virus, Borna disease virus, Ebola virus, new cor
  • the virus may be a plant virus selected from the group consisting of tobacco mosaic virus (TMV), tomato spotted wilt virus (TSWV), cucumber mosaic virus (CMV), potato Y virus (PVY), RT Virus Cauliflower Mosaic Virus (CaMV), Plum Pox Virus (PPV), Brome Mosaic Virus (BMV), Potato Virus X (PVX), Citrus Decay Virus (CTV), Barley Yellow Dwarf Virus (BYDV), Potato Roll Leaf Virus (PLRV), Tomato Cluster Stunt Virus (TBSV), Rice Bulb Virus (RTSV), Rice Yellow Mottle Virus (RYMV), Rice Off-White Virus (RHBV), Maize Reyadofina Virus (MRFV), Maize Dwarf Flower Leaf Virus (MDMV), Sugarcane Mosaic Virus (SCMV), Sweet Potato Feather Mottle Virus (SPFMV), Sweet Potato Settling Vein Nematode Virus (SPSVV),
  • TMV tobacco mosaic
  • examples of bacteria include, but are not limited to, one or more of the following (or a combination thereof): Actinobacillus species, Actinobacillus species, Actinobacillus species, Actinobacillus species ( Actinomyces) species (e.g. Actinomyces israelii and Actinomyces naeslundii), Aeromonas species (e.g.
  • Aeromonas hydrophila Aeromonas veronii biovar soobria (Aeromonas sobria) and Aeromonas los califorans oxidative
  • Anaplasma Anaplasma, Anaplasma, Anaphagocytosis, Anaplasma Actinobacillus actinomycetemcomitans, Bacillus species (e.g. Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis and Bacillus stearothermophilus), Bacteriodes, Enterobacter (Enterobacter) species (e.g. Enterobacter, Enterobacter) Enterobacter cloacae and E. coli, including opportunistic E.
  • enterotoxigenic E. coli such as enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli and uropathogenic E. coli, Enterococcus Species (e.g. Enterococcus faecalis and Enterococcus faecium), Ehrlichia species (e.g.
  • Ehrlichia chafeensia and Ehrlichia canis Epidermophyton floccosum, Erysipelothrix rhusiopathiae, Eubacteria (Francis) species, Francisbacterium obacterium nucleatum, Gardnerella vaginaalis, Gemella morbillorum, Haemophilus species (e.g.
  • Haemophilus influenzae Haemophilusducreyi, Haemophilusaegyptius, Haemophilus parainfluenza, Haemophilus parahaemolyticus, and Haemophilus (parahaemolyticus) (Heoberobacter) cinaedi and Helicobacter fennelliae), Kingella kingii, Klebsiella species, Lactobacillus species, Listeria monocytogenes, Leptospira interrogans, Legionella pneumostophila, Leptospira interrogans, Peptospira interrogans, Peptospira pneumostophila, Leptospira interrogans, Peptospira interrogans, Peptospira species , Microsporum canis, Moraxella catarrhalis, Morganell species, Mobiluncus species, Micrococcus species, Mycobacterium species (e.g.
  • Neisseria gonorrhoeae and Neisseria meningitidis Neisseria gonorrhoeae and Neisseria meningitidis
  • Pasteurella multocida Pityrosporum orbiculare (Malassezia furfur)
  • Providencia species e.g. Providencia alcalifaciens, Providencia rettquifaciens, Providencia rettquifaciens, Providencia rettquifaciens, Providencia stuartiibacterium Pseudomonas aeruginosa), Providencia stuartiibacterium , Rickettsia sp., Salmonella species (e.g.
  • Salmonella enterica Salmonella typhi, Salmonella para typhi, Salmonella enteritidis, Salmonella cholerasuis and Salmonella typhimurium (e.g. Serratia) marquises and Serratia liquices)
  • Shigella species e.g. Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei
  • Staphylococcus species e.g. Streptococcus pneumoniae (e.g.
  • Streptococcus pneumoniae Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae Toxin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, tetracycline-resistant serotype 19F Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, and trimethoprim Serotype 23F Streptococcus pneumoniae, Chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, Spectinomycin-resistant serotype 6B Streptococcus pneumoniae, Streptomycin-resistant serotype 9V Streptococcus pneumoniae, Optokin Resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype
  • target nucleic acid refers to a polynucleotide molecule extracted from a biological sample (sample to be tested).
  • the biological sample is any solid or fluid sample obtained, excreted or secreted from any organism, including but not limited to single-celled organisms, such as bacteria, yeast, protozoa and amoeba, etc., multi-cellular organisms (such as plants or animals, Includes samples from healthy or apparently healthy human subjects or human patients affected by conditions or diseases to be diagnosed or investigated, such as pathogenic microorganisms such as pathogenic bacteria or viral infections).
  • a biological sample can be from, for example, blood, plasma, serum, urine, stool, sputum, mucus, lymphatic fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous fluid, Or any biological fluid obtained from body secretions, exudates, exudates (e.g., fluid obtained from an abscess or any other site of infection or inflammation) or from joints (e.g., normal joints or joints affected by diseases, such as Rheumatoid arthritis, osteoarthritis, gout, or septic arthritis), or a swab on the surface of the skin or mucous membrane.
  • body secretions e.g., exudates, exudates (e.g., fluid obtained from an abscess or any other site of infection or inflammation) or from joints (e.g., normal joints or joints affected by diseases, such as Rheumatoid arthritis, osteoarth
  • the sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or can contain cells (primary cells or cultured cells) or cultured 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 (e.g., blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc. ), tissue biopsy (e.g. tumor biopsy), fine needle aspirate and/or tissue section (e.g. cryostat tissue section and/or paraffin-embedded tissue section).
  • tissue biopsy e.g. tumor biopsy
  • fine needle aspirate and/or tissue section e.g. cryostat tissue section and/or paraffin-embedded tissue section.
  • the biological sample may be plant cells, calluses, tissues or organs (such as roots, stems, leaves, flowers, seeds, fruits) and the like.
  • the target nucleic acid also includes a DNA molecule 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 Technology and non-isothermal amplification technology, isothermal amplification can be based on nucleic acid sequencing 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 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 derivative Material amplification method (RAM).
  • MDA multiple displacement amplification
  • RCA rolling circle amplification
  • LCR ligase chain reaction
  • RAM derivative Material amplification method
  • the detection method of the present invention further includes a step of amplifying the target nucleic acid; the detection system further includes a reagent for amplifying the target nucleic acid.
  • the reagents for amplification include one or more of the following groups: DNA polymerase, strand displacement enzyme, helicase, recombinase, single-stranded binding protein, and the like.
  • Cas protein refers to a CRISPR-associated protein, preferably from a type V CRISPR/CAS protein, once it binds to the characteristic sequence (target sequence) to be detected (ie forms a ternary complex of Cas protein-gRNA-target sequence) ), that is, cutting non-targeted single-stranded nucleic acid detector.
  • target sequence ie forms a ternary complex of Cas protein-gRNA-target 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 activity.
  • the Cis activity refers to the activity of the Cas protein to recognize the PAM site and specifically cleave the target sequence under the action of gRNA.
  • the Cas protein of the present invention includes V-type CRISPR/CAS effector proteins, including protein families such as Cas12, Cas13, and Cas14.
  • Cas12 protein such as Cas12a, Cas1 2b, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j; preferably, the Cas protein is Cas12a, Cas12b, Cas12i, Cas12j.
  • the Cas protein referred to herein also encompasses 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.
  • Functional variants also include fusion products of such a protein with another nucleic acid, protein, polypeptide, or peptide that is not normally related.
  • Functional variants can be naturally occurring or can be man-made.
  • Advantageous embodiments may involve engineered or non-naturally occurring type V DNA targeting effector proteins.
  • one or more nucleic acid molecules encoding Cas proteins, such as Cas12, or orthologs or homologs thereof can be codon optimized for expression in eukaryotic cells. Eukaryotes can be as described herein.
  • the one or more nucleic acid molecules may be engineered or non-naturally occurring.
  • the Cas12 protein or its orthologs or homologs may contain one or more mutations (and therefore the nucleic acid molecule encoding it may have one or more mutations.
  • the mutations may be artificially introduced and may Including but not limited to one or more mutations in the catalytic domain.
  • the Cas protein may be from: Ciliates, Listeria, Corynebacterium, Sartorella, Legionella, Treponema, Nemogen, Eubacterium, Streptococcus , Lactobacillus, Mycoplasma, Bacteroides, Flavivola, Flavobacterium, Sphaerochaeta, nitrogen fixation
  • the Cas protein can be obtained by recombinant expression vector technology, that is, the nucleic acid molecule encoding the protein is constructed on a suitable vector, and then transformed into a host cell, so that the encoding nucleic acid molecule is expressed in the cell, thereby obtaining the corresponding protein.
  • the protein can be secreted by the cell, or the protein can be obtained by cracking the cell through conventional extraction techniques.
  • the coding 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, a 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 includes one or more regulatory elements, selected from the group consisting of promoters, enhancers, ribosome binding sites for translation initiation, terminator, polyadenylic acid sequences, and selection marker genes.
  • the host cell can be a prokaryotic cell, such as Escherichia coli, Streptomyces, Agrobacterium; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell.
  • a prokaryotic cell such as Escherichia coli, Streptomyces, Agrobacterium
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a plant cell.
  • gRNA is also called guide RNA or guide RNA, and has the meaning commonly understood by those skilled in the art.
  • guide RNAs can include direct repeats and guide sequences, or consist essentially of direct repeats and guide sequences (also called spacers in the context of endogenous CRISPR systems). (spacer)) composition.
  • gRNA can include crRNA and tracrRNA, or only crRNA, depending on the Cas protein it depends on.
  • crRNA and tracrRNA can be artificially modified and fused to form single guide RNA (sgRNA).
  • the targeting sequence is any that has sufficient complementarity with the target sequence (the characteristic sequence in the present invention) to hybridize with the target sequence and guide the specific binding of the CRISPR/Cas complex to the target sequence.
  • the polynucleotide sequence usually has a sequence length of 12-25 nt.
  • the same direct repeat sequence can be folded to form a specific structure (such as a stem-loop structure) for the Cas protein to recognize to form a complex.
  • the targeting sequence does not need to be 100% complementary to the characteristic sequence (target sequence).
  • the targeting sequence is not complementary to single-stranded oligonucleotides.
  • the degree of complementarity (match) between the targeting 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 best alignment is within the abilities of those 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 algorithm in matlab, Bowtie, Geneious, Biopython, and SeqMan.
  • the gRNA of the present invention may be natural, or artificially modified or designed and synthesized.
  • the exonuclease or exonuclease described in the present invention means that it can digest a nucleotide strand in double-stranded DNA in a specific direction, that is, hydrolyze a single nucleotide to form a single strand Nucleic acids (also called sticky ends), which include 5'exonuclease and 3'exonuclease.
  • the 5'exonuclease means that it digests the nucleotide chain in the direction of 5' ⁇ 3'
  • the 3'exonuclease means that it digests the nucleotide chain in the direction of 3' ⁇ 5'.
  • the exonuclease of the present invention is preferably a DNA exonuclease.
  • the exonuclease includes but is not limited to: T5 exonuclease, T7 exonuclease, lambda exonuclease or exonuclease VIII and functional
  • the exonuclease of the present invention is preferably an enzyme that does not substantially digest the single-stranded nucleic acid detector.
  • the single-stranded nucleic acid detector of the present invention refers to a sequence containing 2-300 bases, preferably, 3-200, 4-100, 5-50, 5-20 bases.
  • the single-stranded nucleic acid detector may include A, T, C, G, and U nucleotides; in other embodiments, it may also include base modification or may be a nucleic acid analog.
  • the single-stranded nucleic acid detector is used in a detection method or system to report whether it contains a characteristic sequence.
  • the two ends of the single-stranded nucleic acid detector include different reporter groups or labeling molecules. When it is in the initial state (that is, when it is not cut), it does not present a reporter signal.
  • the single-stranded nucleic acid detector is cleaved, it displays A detectable signal is displayed, that is, a detectable difference between after cutting and before cutting is shown. In the present invention, if 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 characteristic sequence to be detected. Sequence of features.
  • 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, Dabcy1 or Tamra.
  • the single-stranded nucleic acid detector has a first molecule (such as FAM or FITC) connected to the 5'end and a second molecule (such as biotin) connected to the 3'end.
  • the reaction system containing the single-stranded nucleic acid detector is matched with the flow bar to detect the characteristic sequence (preferably, the colloidal gold detection method).
  • the flow bar is designed to have two capture lines, the sample contact end (colloidal gold) is provided with an antibody that binds the first molecule (ie, the first molecule antibody), and the first line (control line) contains the binding first molecule.
  • An antibody of one molecule contains an antibody of a second molecule (ie, a second molecule of antibody, such as avidin) that binds to the second molecule at the second line (test line).
  • a second molecule of antibody such as avidin
  • the first molecule of antibody binds to the first molecule and carries the cleaved or uncut single-stranded nucleic acid detector to the capture line, and the cleaved reporter will bind to the first molecule of antibody at the first capture line
  • the uncleaved reporter will bind to the second molecule of antibody at the second capture line.
  • the binding of the reporter group on each line will result in a strong readout/signal (e.g. color).
  • the present invention relates to the use of flow bars as described herein for the detection of nucleic acids.
  • the present invention relates to a method for detecting nucleic acid using a flow strip as defined herein, such as a (lateral) flow test or a (lateral) flow immunochromatographic assay.
  • the molecules in the single-stranded nucleic acid detector can replace each other or change the position of the molecules. As long as the reporting principle is the same or similar to the present invention, the improved methods are also included in the present invention.
  • the detection method of the present invention can be used for the quantitative detection of the characteristic sequence to be detected.
  • the quantitative detection index can be quantified based on the signal strength of the reporter group, for example, based on the luminescence intensity of the fluorescent group, or based on the width of the color band.
  • FIG. 1 Schematic diagram of the principle of the technical solution of the present invention.
  • FIG. 1 The effect of T5 exonuclease on the sensitivity of the Cas12i detection system.
  • line 1 is the control without T5 exonuclease
  • line 2 is the test with T5 exonuclease
  • line 3 is the control without the target nucleic acid.
  • FIG. 7 The effect of T5 exonuclease on the sensitivity of the Cas12a detection system.
  • 1 is the blank control
  • 2 is the experimental group with T5 exonuclease but no target nucleic acid
  • 3 is the experimental group with target nucleic acid but not T5 exonuclease
  • 4 is the simultaneous addition of target nucleic acid and T5 exonuclease Enzyme experimental group.
  • Figure 8 The effect of different exonucleases on the sensitivity of the Cas12b detection system.
  • 1 is the blank control
  • 2 is the experimental group with T5 exonuclease but not the target nucleic acid
  • 3 is the experimental group with T7 exonuclease but not the target nucleic acid
  • 4 is the target nucleic acid but not exonuclease
  • 5 is the experimental group that adds target nucleic acid and T5 exonuclease at the same time
  • 6 is the experimental group that adds target nucleic acid and T7 exonuclease at the same time.
  • a double-stranded target nucleic acid is obtained by an amplification method in a sample to be tested.
  • the target nucleic acid contains a characteristic sequence.
  • suitable primers can be designed according to the characteristic sequence.
  • the double-stranded target nucleic acid will form a single-stranded nucleic acid containing a characteristic sequence;
  • the sequence-paired gRNA guides the Cas protein to recognize and bind to the characteristic sequence; subsequently, the Cas protein stimulates the cleavage activity of the single-stranded nucleic acid detector, which can cut the single-stranded nucleic acid detector in the system; in this embodiment, the single-stranded nucleic acid
  • the two ends of the detector are provided with a fluorescent group and a quenching group respectively. If the single-stranded nucleic acid detector is cleaved, fluorescence will be excited; in other embodiments, the two ends of the single-stranded nucleic acid detector can also be set to be able to Marks detected by colloidal gold.
  • the final concentration of Cas12i (SEQ ID No. 1) in the system is 50 nM
  • the final concentration of gRNA (SEQ ID No. 6) is 50 nM
  • dsDNA double-stranded DNA of target nucleic acid sequence (SEQ ID No. 5)
  • the concentration is 14.8nM
  • the final concentration of single-stranded DNA as Reporter (5'6-FAM-TTTTT-3'BHQ1) is 200nM
  • the concentration of T5 exonuclease is 0.5U/ul.
  • Example 1 Using the target nucleic acid, gRNA, Cas12i and Reporter of Example 1, see Example 1 for the added concentration to verify different exonucleases, including T5 exonuclease, T7 exonuclease and lambda exonuclease, extra-nuclease
  • the added concentration of Dicer is 0.25U/ul, and the samples in different reaction tubes added are as follows:
  • the target nucleic acid and Reporter of Example 1 were used, and the Cas12j (SEQ ID No. 2) protein and gRNA (SEQ ID No. 7) were used; the dosage and concentration of the target nucleic acid, gRNA, and Reporter were 50 nM, 14.8 nM, 50 nM, and 200 nM, respectively.
  • T7 exonuclease has the highest synergistic effect, followed by It is a lambda exonuclease, and T5 exonuclease has the lowest synergistic effect.
  • T5 exonuclease it has a higher synergistic effect than the control without exonuclease.
  • Reporter1 5’-/56-FAM/TGCTTTTTTTCATT/3Bio/-3’;
  • Reporter2 5’-/56-FAM/TTTTT/3Bio/-3’;
  • the detection line of the test strip is labeled with streptavidin that can bind to Bio, and the control line is labeled with an antibody that can bind to a colloidal gold-labeled antibody, and the gold-labeled antibody can bind to FAM.
  • the final concentration of Cas12i is 50nM
  • the final concentration of gRNA is 50nM
  • the concentration of dsDNA (target nucleic acid) is 10nM
  • the final concentration of Reporter is 100nM
  • the added concentration of T5 exonuclease is 0.25U/ul.
  • LAMP was used to amplify the orf1ab gene fragment of SARS-CoV-2, where the LAMP primers were designed as follows:
  • orf1ab-A-B3 agtctgaacaactggtgtaag (SEQ ID NO.: 11);
  • orf1ab-A-BIP tcaacctgaagaagagcaagaactgattgtcctcactgcc (SEQ ID NO.: 12);
  • orf1ab-A-F3 tccagatgaggatgaagaaga (SEQ ID NO.: 13);
  • orf1ab-A-FIP agagcagcagaagtggcacaggtgattgtgaagaagaagag (SEQ ID NO.: 14);
  • orf1ab-A-LB acaaactgttggtcaacaagac (SEQ ID NO.: 15);
  • orf1ab-A-LF ctcatattgagttgatggctca (SEQ ID NO.: 16);
  • the LAMP product Take the LAMP product as the target nucleic acid, use the Cas12i protein of Example 1, and the corresponding gRNA (SEQ ID No. 8); use the single-stranded oligonucleotide 5'-/56-FAM/TTTTT/3Bio/-3' as the target nucleic acid Reporter, by means of lateral flow test strips, adds T5 exonuclease to the system to detect its promoting effect on the sensitivity of the nucleic acid detection system.
  • the detection line of the test strip is marked with streptavidin that can bind to Bio, and the control line is marked with an antibody that can bind to a colloidal gold-labeled antibody, and the gold-labeled antibody can bind to FAM.
  • the final concentration of Cas12i is 50nM
  • the final concentration of gRNA is 50nM
  • the LAMP product is 1ul
  • the reporter concentration is 500nM
  • the dosage of T5 exonuclease is 0.25U/ul.
  • Example 6 Adding T5 exonuclease can improve the detection sensitivity of Cas12a
  • the Cas12a is selected from LbCas12a, and its amino acid sequence is shown in SEQ ID No. 3; in other embodiments, other Cas12a can also be used.
  • the target nucleic acid is OsTGW6 (double-stranded DNA), and the sequence is shown in SEQ ID No. 5.
  • gRNA LbCas12a-TGW6-g1, the sequence is shown in SEQ ID No.9.
  • the Reporter is 5’6-FAM-TTTTT-3’BHQ1.
  • the final concentration of Cas12a is 50 nM
  • the final concentration of gRNA is 50 nM
  • the concentration of dsDNA is 1 nM
  • the final concentration of Reporter single-stranded DNA
  • the concentration of T5 exonuclease is 0.5 U/ul.
  • Example 7 Adding exonuclease can improve the detection sensitivity of Cas12b
  • amino acid sequence of Cas12b is shown in SEQ ID No. 4; in other embodiments, other Cas12b may also be used.
  • the gRNA is AsCas12b-TGW6-g1, and the sequence is shown in SEQ ID No. 10.
  • Example 6 For the concentration of addition, refer to Example 6 to verify the effect of different exonucleases, including T5 exonuclease and T7 exonuclease, on the detection efficiency of Cas12b. As shown in Figure 8, adding different exonucleases can be obvious Improve the sensitivity of Cas12b fluorescence detection and shorten the detection time.
  • Example 8 Cas enzyme can use different single-stranded nucleic acid detectors to detect target nucleic acid
  • single-stranded RNA and nucleic acid analogs are used as single-stranded nucleic acid detectors to verify the effects of different Cas enzymes on target nucleic acid detection.
  • a nucleic acid analog locked nucleic acid is used as a single-stranded nucleic acid detector, and the sequence of the locked nucleic acid (Reporter-LNA) is 5'6-FAM/LNA_T//LNA_T//LNA_T//LNA_T//LNA_T//3'BHQ1;
  • the base of LNA is thymine (T); the results show that Cas12b, Cas12i and Cas12j can show the cleavage activity against the above-mentioned Reporter, and can quickly report fluorescence compared with the control without target nucleic acid.

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Abstract

L'invention concerne un procédé et un système pour effectuer la détection d'un acide nucléique cible à l'aide d'une nouvelle enzyme Cas, et, en particulier, un procédé, un système et un kit pour détecter si une séquence caractéristique à détecter existe ou non dans un acide nucléique cible en utilisant une nouvelle enzyme Cas. Le procédé de détection comprend : l'ajout d'une exonucléase, d'un ARNg, d'une protéine Cas et d'un détecteur d'acide nucléique simple brin dans un système de réaction contenant l'acide nucléique cible. Le procédé peut réduire efficacement le temps de détection et présente une sensibilité plus élevée.
PCT/CN2021/099831 2020-06-17 2021-06-11 Procédé et système pour effectuer une détection d'acide nucléique cible à l'aide d'une nouvelle enzyme cas WO2021254284A1 (fr)

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CN116287130A (zh) * 2023-03-14 2023-06-23 重庆大学 检测谷子抗除草剂基因紧密连锁的分子标记的方法
CN117821630A (zh) * 2024-02-20 2024-04-05 江苏省家禽科学研究所 用于检测肠炎沙门氏菌的核酸检测试剂盒及方法

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019178427A1 (fr) * 2018-03-14 2019-09-19 Arbor Biotechnologies, Inc. Nouveaux systèmes et enzymes de ciblage d'adn crispr
WO2020106630A1 (fr) * 2018-11-19 2020-05-28 The Regents Of The University Of California Procédés de détection et de séquençage d'un acide nucléique cible
CN111235232A (zh) * 2020-01-19 2020-06-05 华中农业大学 基于CRISPR-Cas12a系统的可视化快速核酸检测方法及应用
CN111690717A (zh) * 2020-04-30 2020-09-22 山东舜丰生物科技有限公司 基于crispr技术进行目标核酸检测的方法和系统
CN111690773A (zh) * 2020-06-17 2020-09-22 山东舜丰生物科技有限公司 利用新型Cas酶进行目标核酸检测的方法和系统

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10253365B1 (en) * 2017-11-22 2019-04-09 The Regents Of The University Of California Type V CRISPR/Cas effector proteins for cleaving ssDNAs and detecting target DNAs
CN109055499B (zh) * 2018-08-30 2021-01-19 杭州杰毅生物技术有限公司 基于CRISPR-Cas的等温核酸检测方法及试剂盒
US20210381038A1 (en) * 2018-09-20 2021-12-09 Institute Of Zoology, Chinese Academy Of Sciences Method for detecting nucleic acid
CN113462672A (zh) * 2018-11-15 2021-10-01 中国农业大学 CRISPR-Cas12j酶和系统
CN110029194A (zh) * 2019-04-24 2019-07-19 安邦（厦门）生物科技有限公司 基于CRISPR-Cas基因编辑技术的连续荧光监测检测方法及装置

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019178427A1 (fr) * 2018-03-14 2019-09-19 Arbor Biotechnologies, Inc. Nouveaux systèmes et enzymes de ciblage d'adn crispr
WO2020106630A1 (fr) * 2018-11-19 2020-05-28 The Regents Of The University Of California Procédés de détection et de séquençage d'un acide nucléique cible
CN111235232A (zh) * 2020-01-19 2020-06-05 华中农业大学 基于CRISPR-Cas12a系统的可视化快速核酸检测方法及应用
CN111690717A (zh) * 2020-04-30 2020-09-22 山东舜丰生物科技有限公司 基于crispr技术进行目标核酸检测的方法和系统
CN111690773A (zh) * 2020-06-17 2020-09-22 山东舜丰生物科技有限公司 利用新型Cas酶进行目标核酸检测的方法和系统

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AMAN, R. ET AL.: "Nucleic Acid Detection Using CRISPR/Cas Biosensing Technologies", ACS SYNTH. BIOL, vol. 9, 11 March 2020 (2020-03-11), pages 1226 - 1233, XP055768083, DOI: 10.1021/acssynbio.9b00507 *
HARRINGTON, L.B. ET AL.: "Programmed DNA destruction by miniature CRISPR-Casl4 enzymes", SCIENCE, vol. 362, 16 November 2018 (2018-11-16), pages 839 - 842, XP055614750, DOI: 10.1126/science.aav4294 *
YAN, W.X. ET AL.: "Functionally diverse type V CRISPR-Cas systems", SCIENCE, vol. 363, 6 December 2018 (2018-12-06), pages 88 - 91, XP055594948, DOI: 10.1126/science.aav7271 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115125313A (zh) * 2022-05-31 2022-09-30 吉林大学 一种致病性小肠结肠炎耶尔森氏菌检测用引物对和CRISPR/Cas12a检测试剂盒
CN116287130A (zh) * 2023-03-14 2023-06-23 重庆大学 检测谷子抗除草剂基因紧密连锁的分子标记的方法
CN117821630A (zh) * 2024-02-20 2024-04-05 江苏省家禽科学研究所 用于检测肠炎沙门氏菌的核酸检测试剂盒及方法

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