CN111733216A - Method for improving detection efficiency of target nucleic acid - Google Patents

Method for improving detection efficiency of target nucleic acid Download PDF

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CN111733216A
CN111733216A CN202010576290.XA CN202010576290A CN111733216A CN 111733216 A CN111733216 A CN 111733216A CN 202010576290 A CN202010576290 A CN 202010576290A CN 111733216 A CN111733216 A CN 111733216A
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CN111733216B (en
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梁亚峰
段志强
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Shandong Shunfeng Biotechnology Co Ltd
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Abstract

The invention provides a method for improving the detection efficiency of target nucleic acid, and particularly relates to a method for improving the detection efficiency of target nucleic acid based on a CRISPR technology. The method can have higher sensitivity.

Description

Method for improving detection efficiency of target nucleic acid
Technical Field
The invention relates to the field of nucleic acid detection, in particular to a method for improving the detection efficiency of target nucleic acid, and specifically relates to a method for improving the detection efficiency of target nucleic acid based on a CRISPR technology.
Background
The method for specifically detecting Nucleic acid molecules (Nucleic acid detection) has important application values, such as pathogen detection, genetic disease detection and the like. In the aspect of pathogen detection, each pathogenic microorganism has a unique characteristic nucleic acid molecule sequence, so that nucleic acid molecule detection for a specific species, also called Nucleic Acid Diagnostics (NADs), can be developed, and is important in the fields of food safety, detection of environmental microbial contamination, infection of human pathogenic bacteria, and the like. 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 genomic level provides a new perspective for modern molecular biology, and SNPs are closely related to biological functions, evolution, diseases and the like, so the development of detection and analysis techniques of SNPs is particularly important.
The detection of specific nucleic acid molecules established today usually requires two steps, the first step being the amplification of the nucleic acid of interest and the second step being the detection of the nucleic acid of interest. The existing detection technologies include restriction endonuclease methods, Southern, Northern, dot blot, fluorescent PCR detection technologies, LAMP loop-mediated isothermal amplification technologies, recombinase polymerase amplification technologies (RPA) and the like. After 2012, CRISPR gene editing technology arose, a new nucleic acid diagnosis technology (SHERLOCK technology) of targeted RNA with Cas13 as a core was developed by the zhanfeng team based on RPA technology, a diagnosis technology (DETECTR technology) with Cas12 enzyme as a core was developed by the Doudna team, and a new nucleic acid detection technology (HOLMES technology) based on Cas12 was also developed by the royal doctor of the institute of physiology and ecology of plants in the shanghai of the chinese academy of sciences. Nucleic acid detection techniques developed based on CRISPR technology are playing an increasingly important role.
Despite the numerous existing nucleic acid detection technologies, how to perform faster, easier, cheaper, and more accurate detection is still an important direction for improving the detection technology. Therefore, the development of novel detection systems and detection methods is still of great significance in the field of nucleic acid detection.
Disclosure of Invention
The invention provides a method, a system and a kit for detecting target nucleic acid based on CRISPR technology.
In one aspect, the invention provides a high-sensitivity method for detecting whether a characteristic sequence to be detected exists in a target nucleic acid based on a CRISPR technology, comprising the following steps:
(1) providing target nucleic acid, nickase, strand displacement DNA polymerase, dNTPs, gRNA, Cas protein and a single-stranded nucleic acid detector, wherein the target nucleic acid is provided with an enzyme cutting recognition site of the nickase;
(2) the nickase cuts the enzyme cutting recognition site to form a gap, and the strand displacement DNA polymerase extends from the gap to displace at least one single-stranded nucleic acid;
(3) the gRNA can target a characteristic sequence to be detected, the Cas protein can recognize the characteristic sequence to be detected under the action of the gRNA, and the Cas protein can cut the single-stranded nucleic acid detector;
(4) the single-stranded nucleic acid detector exhibits a detectable signal after cleavage by the Cas protein compared to the single-stranded nucleic acid detector before cleavage by the Cas protein;
(5) testing whether the detectable signal of step (4) can be detected; if the detectable signal in the step (4) can be detected, reflecting that the target nucleic acid contains the characteristic sequence to be detected; alternatively, if the detectable signal in step (4) is not detected, it is reflected that the target nucleic acid does not contain the signature sequence to be detected.
According to the invention, because the target nucleic acid is provided with the enzyme cutting recognition site of the nickase, the single-stranded nucleic acid can be replaced by taking the target nucleic acid as a template under the action of the nickase and the strand displacement DNA polymerase; if the target nucleic acid contains the characteristic sequence to be detected, a large amount of single-stranded nucleic acid containing the characteristic sequence to be detected can be obtained by displacement under the action of nicking enzyme and strand displacement DNA polymerase. The Cas protein can recognize a characteristic sequence to be detected in a target nucleic acid, and can also recognize a characteristic sequence to be detected in a replaced single-stranded nucleic acid. Upon recognition of the signature sequence to be detected, the Cas protein is able to cleave the added single-stranded nucleic acid detector and thereby exhibit a detectable signal. The present inventors have found that the sensitivity of the detection can be greatly improved as compared with the control group by adding a nicking enzyme and a strand displacement DNA polymerase.
In a preferred embodiment, the method further comprises the step of adding a single-chain binding protein (SSB) which further increases the sensitivity of the detection.
The single-chain binding protein comprises Escherichia coli SSB, T4 gp32, T7 gp2.5, RecA, or a combination thereof.
Further, the Cas protein is a protein having trans cleavage activity, preferably, a V-type CRISPR/Cas effector protein.
More preferably, the V-type Cas protein is selected from any one or any combination of Cas12 and Cas14 family proteins; preferably, the Cas12 family protein is selected from one or any combination of Cas12i, Cas12j, Cas12a, Cas12b, Cas12d, Cas12e, Cas12f, Cas12g and Cas12 h; more preferably, the Cas12 family protein is one or a combination of any two of Cas12i, Cas12j, Cas12a and Cas12 b.
In the invention, the nicking enzyme is a tool enzyme which can recognize a DNA double strand but only cut a single strand of a recognition site, and preferably, the nicking enzyme is one or more of Nb.BbCI, Nb.Bsml, Nb.BsrDI, Nb.BssSI, NbBstI, Nt.AlwI, Nt.BbCI, Nt.BsmAl, Nt.BspQl or Nt.BstNBl;
more preferably, the nicking enzyme is selected from nb.
In the present invention, the strand displacement DNA polymerase is selected from Klenow Fragment (3 '-5' exo)-) Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, TherminatorTMOne or more of DNA polymerase and Phi29DNA polymerase, preferably Klenow fragment (3 '-5' exo)-)。
In the present invention, the detectable signal can be realized by: vision-based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization, colloidal phase transition/dispersion, electrochemical detection, and semiconductor-based detection.
In the present invention, the detectable signal may 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 may be read by any suitable means, including but not limited to: measurement of a detectable fluorescent signal, gel electrophoresis detection (by detecting a change in a band on the gel), detection of the presence or absence of a color based on vision or a sensor, or a difference in the presence of a color (e.g., based on gold nanoparticles) and a difference in an electrical signal.
In one embodiment, the 5 'end and the 3' end of the single-stranded nucleic acid detector are respectively provided with different reporter groups, and when the single-stranded nucleic acid detector is cut, the single-stranded nucleic acid detector can show a detectable reporter signal, and whether the target nucleic acid contains the characteristic sequence to be detected is reflected by the existence of the reporter signal;
alternatively, in other embodiments, the 5 'end and the 3' end of the single-stranded nucleic acid detector are respectively provided with different labeled molecules, and the results of the single-stranded nucleic acid detector before and after cleavage by the Cas protein are detected by a lateral flow test strip, for example, a colloidal gold test strip, to reflect whether the target nucleic acid contains the characteristic sequence to be detected.
In one embodiment, the target nucleic acid comprises DNA, RNA, preferably double-stranded nucleic acid, single-stranded nucleic acid, more preferably double-stranded DNA.
In one embodiment, the target nucleic acid is derived from a sample of a virus, bacterium, microorganism, soil, water source, human, animal, plant, or the like.
In a preferred embodiment, the target nucleic acid is obtained by amplifying a primer, and in a preferred embodiment, the primer is provided with an enzyme cutting recognition site of the nicking enzyme. Thus, the amplified target nucleic acid contains a cleavage recognition site for a nicking enzyme at the 5 'end and/or the 3' end. Preferably, the target nucleic acid is the product of PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM amplification.
In one embodiment, the method further comprises the step of obtaining the target nucleic acid from the sample.
In one embodiment, the characteristic sequence to be detected is a virus-specific sequence, a bacteria-specific sequence, a characteristic sequence associated with a disease, a specific mutation site or an SNP site; preferably, the virus is a plant virus or an animal virus; preferably, the virus is a coronavirus, preferably SARS, SARS-CoV2(COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-CoV. If the target nucleic acid has the characteristic sequence to be detected, the sample from which the target nucleic acid is derived can be reflected to be a certain virus, a certain bacterium, or infected with a certain virus, a certain bacterium, or a certain disease, or to have a specific mutation site or SNP site.
In one embodiment, the Cas protein is a protein having double-stranded and/or single-stranded cleavage activity.
In one embodiment, the Cas protein is a protein having cis and trans cleavage activity.
In one embodiment, the Cas protein is selected from the group consisting of a type V CRISPR/Cas effector protein comprising: cas12, Cas14 family proteins, or mutants thereof.
In one embodiment, the Cas protein mutant comprises amino acid substitutions, deletions or substitutions, and the mutant retains at least its trans cleavage activity. Preferably, the mutant has cis and trans cleavage activity.
In one embodiment, the Cas protein is preferably a Cas12 family, including but not limited to Cas12a, Cas12b, Cas12i, Cas12 j;
in one embodiment, the Cas12a is selected from one of FnCas12a, assas 12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12 a; the Cas12a is preferably LbCas12a, the amino acid sequence is shown as SEQ ID No.3, or the derivative protein which is formed by substituting, deleting or adding one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues of the amino acid sequence shown as SEQ ID No.3 or an active fragment thereof and has basically the same function.
In other embodiments, the amino acid sequence of Cas12b is shown in SEQ ID No.4, or a derivative protein formed by substitution, deletion, or addition of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid residues of the amino acid sequence shown in SEQ ID No.4 or an active fragment thereof, and having substantially the same function.
In preferred embodiments, the amino acid sequence of the Cas12i protein is selected from the group consisting of:
(1) SEQ ID NO: 1;
(2) converting SEQ ID NO: 1 or an active fragment thereof by substitution, deletion or addition of one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues, and has substantially the same function.
The amino acid sequence of the Cas12j protein is selected from the group consisting of:
(1) SEQ ID NO: 2;
(2) converting SEQ ID NO: 2 or an active fragment thereof by substitution, deletion or addition of one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues, and has substantially the same function.
In one embodiment, the Cas protein mutant comprises amino acid substitutions, deletions or substitutions, and the mutant retains at least its trans cleavage activity. Preferably, the mutant has Cis and trans cleavage activity.
In one embodiment, the gRNA comprises a sequence targeting the signature sequence to be detected (a guide sequence) and a sequence recognizing the Cas protein (a direct repeat or a portion thereof).
In one embodiment, the targeting sequence comprises 10-40 bp; preferably, 12-25 bp; preferably, 15-23 bp; preferably, 16-18 bp.
In one embodiment, the gRNA has at least a 50% match to the signature sequence to be detected, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.
In one embodiment, when the signature sequence contains one or more signature sites (e.g., a particular mutation site or SNP), the signature site is a perfect match to the gRNA.
In one embodiment, one or more grnas targeting different sequences may be included in the detection method, targeting different signature sequences.
In one embodiment, said identifying said feature sequence to be detected comprises binding and/or cleaving the feature sequence to be detected.
In one embodiment, the steps (4) and (5) may be implemented by: the 5 'end and the 3' end of the single-stranded nucleic acid detector are respectively provided with different reporter groups, when the single-stranded nucleic acid detector is cut, a detectable reporter signal can be shown, and whether the target nucleic acid contains a characteristic sequence to be detected is reflected by the existence of the reporter signal; if the reporter signal can be detected, the target nucleic acid contains the characteristic sequence to be detected; alternatively, if the reporter signal is not detectable, it is indicative that the target nucleic acid does not contain the signature sequence to be detected. For example, two ends of the single-stranded nucleic acid detector are respectively provided with a fluorescent group and a quenching group, when the single-stranded nucleic acid detector is cut, a detectable fluorescent signal can be shown, and whether the target nucleic acid contains the characteristic sequence to be detected is reflected by the existence of the fluorescent signal; the fluorescent signal can be detected, and the target nucleic acid contains a characteristic sequence to be detected; alternatively, if the fluorescent signal is not detected, it indicates that the target nucleic acid does not contain the signature sequence to be detected.
In one embodiment, the fluorescent group is selected from one or any of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC Red 460; the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.
In one embodiment, the steps (4) and (5) can also be realized by other ways: the 5 'end and the 3' end of the single-stranded nucleic acid detector are respectively provided with different marker molecules, and the test results of the colloidal gold test strip before the single-stranded nucleic acid detector is cut by the Cas protein and after the single-stranded nucleic acid detector is cut by the Cas protein are detected in a colloidal gold detection mode so as to reflect whether the target nucleic acid contains a characteristic sequence to be detected; the single-stranded nucleic acid detector shows different color development results on the detection line and the quality control line of the lateral flow test strip before and after being cut by the Cas protein.
In one embodiment, the Cas protein and gRNA are used in a molar ratio of (0.8-1.2): 1.
in one embodiment, the Cas protein is used in a final concentration of 20-200nM, preferably, 30-100nM, more preferably, 40-80nM, more preferably, 50 nM.
In one embodiment, the gRNA is used in a final concentration of 20-200nM, preferably, 30-100nM, more preferably, 40-80nM, and more preferably, 50 nM.
In one embodiment, the target nucleic acid is used in a final concentration of 5-100nM, preferably, 10-50 nM.
In one embodiment, the single stranded nucleic acid detector is used at a final concentration of 100-.
In one embodiment, the nicking enzyme is used in a final concentration of 0.1-5.0U/. mu.l, preferably, 0.2-3.0U/. mu.l, more preferably, 0.3-2.0U/. mu.l, more preferably, 0.5-1.0U/. mu.l.
The strand displacement DNA polymerase is used in a final concentration of 0.05 to 5.0U/. mu.l, preferably 0.1 to 3.0U/. mu.l, more preferably 0.1 to 2.0U/. mu.l, more preferably 0.1 to 1.0U/. mu.l.
The final dosage concentration of the SSB is 0.1ng/ul-5ng/ul, preferably 0.2ng/ul-3ng/ul, and more preferably 0.4ng/ul-2 ng/ul.
The dNTPs are used in a final concentration of 0.1-5mM, preferably, 0.2-3mM, more preferably, 0.5-2 mM.
In one embodiment, the single stranded nucleic acid detector has 2 to 200 bases, preferably 3 to 100 bases, preferably 3 to 30 bases, preferably 4 to 20 bases, more preferably 5 to 15 bases.
In the present invention, the single-stranded nucleic acid detector includes a single-stranded DNA, a single-stranded RNA, or a single-stranded DNA-RNA hybrid. In other embodiments, the single-stranded nucleic acid detector comprises a mixture of any two or three of single-stranded DNA, single-stranded RNA, or single-stranded DNA-RNA hybrids, e.g., a combination of single-stranded DNA and single-stranded RNA, a combination of single-stranded DNA and single-stranded DNA-RNA hybrids, and a combination of single-stranded RNA and single-stranded DNA-RNA.
In other embodiments, the single stranded nucleic acid detector further comprises a nucleic acid modification or a nucleic acid analog; such as base modifications, backbone modifications, sugar modifications, and the like, to provide new or enhanced features (e.g., improved stability) to the nucleic acid. Examples of suitable modifications include modified nucleic acid backbones and non-natural internucleoside linkages, and nucleic acids having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Suitable modified oligonucleotide backbones containing phosphorus atoms therein include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates. Suitable types of base modifications include one or more of deamination, methylation, acetylation, hydrogenation, fluorination, or sulfurization modifications. In some embodiments, the single stranded nucleic acid detector comprises one or more phosphorothioate and/or heteroatomic nucleotide linkages.
Suitable nucleic acid analogs include, but are not limited to: 2 '-O-methyl substituted RNA, locked nucleic acid, bridged nucleic acid, morpholine nucleic acid, ethylene 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' -thio RNA and combinations thereof in one embodiment, the method can be used for the quantitative detection of the signature sequence to be detected.
In another aspect, the present invention also provides a system for detecting whether a characteristic sequence to be detected exists in a target nucleic acid based on CRISPR technology, the system comprising: nicking enzyme, strand displacement DNA polymerase, dNTPs, gRNA, Cas protein and single-stranded nucleic acid detector; the target nucleic acid is provided with an enzyme cutting recognition site of the nickase;
wherein the nicking enzyme cleaves the enzyme cleavage recognition site to form a nick, and the strand displacement DNA polymerase extends from the nick to displace at least one single-stranded nucleic acid;
the gRNA can target a 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 excites the cleavage activity of a single-stranded nucleic acid detector after recognizing the characteristic sequence to be detected;
the single-stranded nucleic acid detector exhibits a detectable signal after cleavage by the Cas protein compared to the single-stranded nucleic acid detector before cleavage by the Cas protein;
if the detectable signal can be detected, the target nucleic acid contains the characteristic sequence to be detected; alternatively, if the detectable signal is not detectable, it is indicative that the target nucleic acid does not contain the signature sequence to be detected.
In preferred embodiments, the system further comprises a single-chain binding protein.
In another aspect, the present invention also provides a kit for detecting the presence or absence of a characteristic sequence to be detected in a target nucleic acid based on CRISPR technology, the kit comprising: nicking enzyme, strand displacement DNA polymerase, dNTPs, gRNA, Cas protein and single-stranded nucleic acid detector.
In a preferred embodiment, the kit further comprises a single-chain binding protein.
Further, the kit also comprises a primer for amplifying the target nucleic acid. Preferably, the primer is provided with an enzyme cutting recognition site of the nicking enzyme, so that the amplified target nucleic acid is provided with the enzyme cutting recognition site of the nicking enzyme.
In another aspect, the invention also provides the use of the system or the kit in diagnosing whether the characteristic sequence to be detected exists in a sample to be detected.
Further, the use comprises obtaining target nucleic acid from a sample to be detected, and further detecting whether the characteristic sequence to be detected exists in the target nucleic acid.
Preferably, the target nucleic acid may be obtained from the sample to be tested by nucleic acid sequencing-based amplification (NASBA), Recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), Strand Displacement Amplification (SDA), helicase-dependent amplification (HDA), or Nicking Enzyme Amplification Reaction (NEAR), PCR, Multiple Displacement Amplification (MDA), Rolling Circle Amplification (RCA), Ligase Chain Reaction (LCR), or derivative amplification methods (RAM).
In a preferred embodiment, the characteristic sequence to be detected is a virus-specific sequence, a bacteria-specific sequence, a characteristic sequence related to a disease, a specific mutation site or an SNP site; preferably, the virus is a plant virus or an animal virus; preferably, the virus is a coronavirus, preferably SARS, SARS-CoV2(COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-CoV. If the target nucleic acid has the characteristic sequence to be detected, the sample from which the target nucleic acid is derived can be reflected to be a certain virus, a certain bacterium, or infected with a certain virus, a certain bacterium, or a certain disease, or to have a specific mutation site or SNP site.
General definition:
unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
The term "amino acid" refers to a carboxylic acid containing an amino group. Each protein in an organism is composed of 20 basic amino acids.
The terms "polynucleotide", "nucleotide sequence", "nucleic acid molecule" and "nucleic acid" are used interchangeably and include DNA, RNA or hybrids thereof, whether double-stranded or single-stranded.
The term "homology" or "identity" is used to refer to a sequence between two polypeptides or between two nucleic acids
And (4) matching the situation. When two sequences to be compared are substituted by the same base or amino acid monomer at a position
When a subunit is occupied (e.g., a position in each of two DNA molecules is occupied by adenine,
or a position in each of the two polypeptides is occupied by a lysine), then the respective molecule is at that position
Are identical. Between the two sequences. Typically, this is done when the two sequences are aligned to yield maximum identity
And (6) comparing. Such an alignment can be determined by using, for example, the identity of the amino acid sequences by conventional methods, as taught by, for example, Smith and Waterman,1981, adv.Appl.Math.2:482Pearson & Lipman,1988, Proc.Natl.Acad.Sci.USA 85:2444, Thompson et al, 1994, Nucleic Acids Res 22:467380, etc., by computerized operational 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 /), can also be used, determined using default parameters.
As used herein, the "CRISPR" refers to clustered, regularly interspaced short palindromic repeats (clustered regularly interspaced short palindromic repeats) derived from the immune system of a microorganism.
As used herein, "biotin", also known as vitamin H, is a small molecule vitamin with a molecular weight of 244 Da. "avidin", also called avidin, is a basic glycoprotein having 4 binding sites with extremely high affinity to biotin, and streptavidin is a commonly used avidin. The very strong affinity of biotin to avidin can be used to amplify or enhance the detection signal in the detection system. For example, biotin is easily bonded to a protein (such as an antibody) by a covalent bond, and an avidin molecule bonded to an enzyme reacts with a biotin molecule bonded to a specific antibody, so that not only is a multi-stage amplification effect achieved, but also color is developed due to the catalytic effect of the enzyme when the enzyme meets a corresponding substrate, and the purpose of detecting an unknown antigen (or antibody) molecule is achieved.
Characteristic sequence
As used herein, the terms "signature sequence" or "signature sequence to be detected" are used interchangeably and refer to a nucleic acid sequence that characterizes an organism-specific or certain characteristic feature that hybridizes to a gRNA guide sequence to promote formation of a CRISPR complex. The signature sequence is a DNA polynucleotide, which can comprise a portion complementary to the gRNA guide sequence in an amount that is the same as or slightly less than the portion complementary to the gRNA guide sequence. In certain embodiments the organisms include animals, plants and microorganisms. The microorganism includes bacteria, fungi, yeast, protozoa, parasites or viruses. For example, the signature sequence may be a nucleic acid sequence which characterises the virus (including a DNA sequence formed by reverse transcription if the virus is an RNA sequence); for example, the signature sequence may be a sequence containing a specific mutation site, such as a tumor-inducing gene mutation site in an animal cell, or some gene mutation site that alters a plant trait in a plant (e.g., a specific mutation site that confers herbicide resistance to an ALS protein).
In certain embodiments, the virus comprises a double-stranded RNA virus, a positive-sense RNA virus, a negative-sense RNA virus, a retrovirus, or a combination thereof, or the viral infection is caused by a virus of the family Coronaviridae (Coronaviridae), Picornaviridae (Picornaviridae), Caliciviridae (Caliciviridae), Flaviviridae (Flaviviridae), Togaviridae (Togaviridae), Filoviridae (Filoviridae), Paramyxoviridae (Paramyxoviridae), Pneumoviridae (Pneumoviridae), Rhabdoviridae (Rhabdoviridae), Arenaviridae (Arenaviridae), Bunyaviridae (Bunyaviridae), Orthomyxoviridae (Orthomyxoviridae), or Delta viruses, or the viral infection is caused by a coronavirus (Corona virus), Rhinovirus (Rhinovirus), Rhinovirus (Rhinovirus a), Rhinovirus (Rhinovirus), Rhinovirus (Rhinovirus a), Rhinovirus (Rhinovirus), Rhinovirus (Rhinovirus a), Rhinovirus (Rhinovirus), Rhinovirus (Rhinovirus) or a), Rhinovirus (Rhinovirus) infection by a), Rhinovirus (Rhinovirus) or a), Hepatitis C virus (Hepatitis C virus), Dengue virus (Dengue River virus), Zikavirus (Zikavirus), Rubella virus (Rubella virus), Ross River virus (Ross River virus), Sindbis virus (Sindbis virus), Chikungunya virus (Chikungunya virus), Borna disease virus (Borna disease virus), Ebola virus (Ebola virus), novel coronavirus (2019-nCoV), Marburg virus (Marburg virus), measles virus (Measles virus), Mumps virus (Mumps virus), Nipah virus (Nipah virus), Hendra virus (Hendra virus), Newcastle disease virus (Newcastle disease virus), human respiratory syncytial virus (Humanrespiratory syncytial virus), Rabies virus (rabis virus), Lassa virus (Lassa virus), Hantavirus (Hantavirus), Crimean-Congo hemorrhagic fever virus (Crimean-conmorrharge virus), Influenza (inflenza), or Hepatitis D virus (hepatis D virus).
In certain exemplary embodiments, 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 Virus Y (PVY), RT virus cauliflower mosaic virus (CaMV), plum blossom pox virus (PPV), Brome Mosaic Virus (BMV), Potato Virus X (PVX), Citrus Tristeza Virus (CTV), Barley Yellow Dwarf Virus (BYDV), potato leafroll virus (PLRV), tomato clumping trick virus (TBSV), rice corm virus (RTSV), Rice Yellow Mottle Virus (RYMV), rice grey white virus (RHBV), maize raleigh phenanthroline virus (MRFV), Maize Dwarf Mosaic Virus (MDMV), sugarcane mosaic virus (SCMV), sweet potato feather mottle virus (SPMV), sweet potato sedimentary vein nematode virus (SPV), grape flabellum virus (GFLV), Grape Virus A (GVA), Grape Virus B (GVB), grape spotted virus (GFkV), Grape leaf curl virus-related viruses-1, -2, and-3, (GLRaV-1, -2, and-3), arabis mosaic virus (ArMV), or larch numb-locus-related virus (RSPaV).
In certain embodiments, examples of bacteria include, but are not limited to, one or more (or a combination of) the following: actinobacillus (Actinobacillus), Actinomycetes (Actinomycetes), Actinomycetes (Actinomyces), Aeromonas (Aeromonas) such as Aeromonas hydrophylla, Aeromonas campestris and Aeromonas campestris, Anaplasia angularis, Anaplasia marcescens Alcaligenes Xyloxifragans, Acetobacter baumii, Actinomyces Actinomycetes, Escherichia coli, Fusobacterium nuclearum, Gardnerella vagenalis, Gemelalla Morblillum, Haemophilus (Haemophilus) species (e.g.Haemophilus influezae, Haemophilus ducreyi, Haemophilus saegypticus, Haemophilus parahaemophilus parainfluenzae, Haemophilus Haemophilus and Haemophilus parahaemolyticus, Helicobacter (e.g.Helicobacter pararhizophilus, Helicobacter cinalis and Helicobacter fennellae), Kingella kingii, Klebsiella (Klebsiella) species, Lactobacillus (Lactobacillus) species, Listeria monocytogenes, Leptomonas, Lactobacillus (Lactobacillus) species, Corynebacterium, Lactobacillus species, Mycobacterium species, such as strains, strains such as strains of Bacillus species of Bacillus, strains of Bacillus (Lactobacillus species such as strains of Bacillus, strains of Bacillus, strains of Bacillus, strains of Bacillus, strains of strains, Pasteurella multocida, Pityrosporum orbiculare (Malassezia furur), Providence sp (Providence), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Rickettsia sp, Salmonella (Salmonella) species (Salmonella enterica, Salmonella typhi, Salmonella serotype pauli, Salmonella enteritidis, Salmonella choleraesuis and Salmonella typhimurium), Serratia (Serratia) species (such as Serratia pneumoniae and Serratia aliella serotype), Shigella (Shigella) and Shigella typhimurium), Serratia (Serratia) species (such as Serratia pneumoniae and Shigella serotype Shigella), Shigella (Shigella) resistant (Shigella resistant) species (such as Shilaseriella pneumoniae, Shigella resistant) such as Shigella, Shigella resistant S (Shigella), Shigella (Shigella) species (Shigella) resistant such as Shigella resistant, Shigella resistant (Shigella) s Streptococcus pneumoniae and Shigella streptococci) such as Shigella serotype S pneumoniae, Shigella resistant (Shigella resistant) and Shigella resistant (Shigella resistant) such as Shigella serotype V, Shigella resistant (Shigella resistant) such as Shigella serotype 4, Shigella resistant (Shigella resistant) and Shigella resistant (Shigella resistant) such as Shigella resistant) and Shigella resistant (Shigella resistant) such as Shigella resistant) s resistant (Shigella resistant) such as Shigella resistant, Rifampin-resistant serotype 18C streptococcus pneumoniae, tetracycline-resistant serotype 19F streptococcus pneumoniae, penicillin-resistant serotype 19F streptococcus pneumoniae, and trimethoprim-resistant serotype 23F streptococcus pneumoniae, chloramphenicol-resistant serotype 4 streptococcus pneumoniae, spectinomycin-resistant serotype 6B streptococcus pneumoniae, streptomycin-resistant serotype 9V streptococcus pneumoniae, omphosin-resistant serotype 14 streptococcus pneumoniae, rifampin-resistant serotype 18C streptococcus pneumoniae, penicillin-resistant serotype 19F streptococcus pneumoniae, or trimethoprim-resistant serotype 23F streptococcus pneumoniae), Yersinia pestis (Yersinia) species (e.g., Yersinia entericotica, Yersinia pestis, and Yersinia pseudoticus), and xanthos maltophilia, among others.
Target nucleic acid
As used herein, the "target nucleic acid" refers to a polynucleotide molecule extracted from a biological sample (a 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, yeasts, protozoa and amoebae and the like, multicellular organisms (e.g. plants or animals, including samples from healthy or superficially healthy human subjects or human patients affected by a condition or disease to be diagnosed or investigated, e.g. infection by a pathogenic microorganism such as a pathogenic bacterium or virus). For example, the biological sample may be a biological fluid 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 humor, or any bodily secretion, exudate (e.g., obtained from an abscess or any other site of infection or inflammation), or a fluid obtained from a joint (e.g., a normal joint or a joint affected by a disease, such as rheumatoid arthritis, osteoarthritis, gout, or septic arthritis), or a swab of a skin or mucosal surface. The sample may also be a sample obtained from any organ or tissue (including biopsies or autopsy specimens, e.g., tumor biopsies) or may comprise cells (primary cells or cultured cells) or culture medium conditioned by any cell, tissue or organ. Exemplary samples include, but are not limited to, cells, cell lysates, blood smears, cytocentrifuge preparations, cytological smears, bodily fluids (e.g., blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.), tissue biopsies (e.g., tumor biopsies), fine needle aspirates, and/or tissue sections (e.g., cryostat tissue sections and/or paraffin-embedded tissue sections).
In other embodiments, the biological sample may be a plant cell, callus, tissue or organ (e.g., root, stem, leaf, flower, seed, fruit), and the like.
In the present invention, the target nucleic acid also includes a DNA molecule formed by reverse transcription of RNA, and further, the target nucleic acid may be amplified by a technique known in the art, such as isothermal amplification techniques, such as 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 (NEAR), and non-isothermal amplification techniques. In certain exemplary embodiments, 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 amplification methods (RAM).
Further, the detection method of the present invention further comprises a step of amplifying the target nucleic acid; the detection system further comprises a reagent for amplifying the target nucleic acid. The reagents for amplification include one or more of the following: DNA polymerase, strand displacing enzyme, helicase, recombinase, single-strand binding protein, and the like.
Cas protein
As used herein, "Cas protein" refers to a CRISPR-associated protein, preferably from type V CRISPR/Cas protein, that, once bound to a signature sequence (target sequence) to be detected (i.e., a ternary complex of Cas protein-gRNA-target sequence is formed), can induce its trans activity, i.e., random cleavage of non-targeted single-stranded nucleic acids (i.e., the single-stranded nucleic acid detector described herein, e.g., single-stranded DNA, and/or single-stranded RNA, and/or single-stranded DNA-RNA hybrids, preferably single-stranded oligonucleotides). When the Cas protein is combined with the characteristic sequence, the protein can induce the trans activity by cutting or not cutting the characteristic sequence; preferably, it induces its trans activity by cleaving the signature sequence; more preferably, it induces its trans activity by cleaving the single-stranded signature sequence.
The Cas protein of the present invention is a protein having at least trans cleavage activity, preferably, the Cas protein is a protein having cis and trans cleavage activity. The cis activity refers to the activity of the Cas protein in recognizing a target sequence and specifically cutting the target sequence under the action of the gRNA.
The Cas protein provided by the invention comprises V-type CRISPR/CAS effector proteins, including protein families such as Cas12 and Cas 14. Preferably, e.g., Cas12 proteins, e.g., Cas12a, Cas12b, Cas12i, Cas12 j; preferably, the Cas protein is Cas12a, Cas12i, Cas12 j.
In embodiments, a Cas protein, as referred to herein, such as Cas12, also encompasses a functional variant of Cas or a homolog or ortholog thereof. As used herein, a "functional variant" of a protein 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 fusion products of such proteins with another, usually unrelated, nucleic acid, protein, polypeptide or peptide. Functional variants may be naturally occurring or may be artificial. Advantageous embodiments may relate to engineered or non-naturally occurring V-type DNA targeting effector proteins.
In one embodiment, one or more nucleic acid molecules encoding a Cas protein, such as Cas12, or orthologs or homologs thereof, may be codon optimized for expression in a eukaryotic cell. Eukaryotes can be as described herein. One or more nucleic acid molecules may be engineered or non-naturally occurring.
In one embodiment, the Cas12 protein or ortholog or homolog thereof may comprise one or more mutations (and thus the nucleic acid molecule encoding it may have one or more mutations.
In one embodiment, the Cas protein may be from: cilium, listeria, corynebacterium, satt's, legionella, treponema, Proteus, eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flavivivola, Flavobacterium, Sphaerochaeta, Azospirillum, gluconacetobacter, Neisseria, Rochelia, Parvibaculum, Staphylococcus, Nitratifroctor, Mycoplasma, Campylobacter, and Muspirillum.
In one embodiment, the Cas protein further includes proteins having 50%, preferably 55%, preferably 60%, preferably 65%, preferably 70%, preferably 75%, preferably 80%, preferably 85%, preferably 90%, preferably 95%, sequence identity to the above sequences and having trans activity.
The Cas protein can be obtained by recombinant expression vector technology, namely, a nucleic acid molecule encoding the protein is constructed on a proper vector and then is transformed into a host cell, so that the encoding nucleic acid molecule is expressed in the cell, and the corresponding protein is obtained. The protein can be secreted by cells, or the protein can be obtained by breaking cells through a conventional extraction technology. The encoding nucleic acid molecule may or may not be integrated into the genome of the host cell for expression. The vector may further comprise regulatory elements which facilitate sequence integration, or self-replication. The vector may be, for example, of the plasmid, virus, cosmid, phage, etc. type, which are well known to those skilled in the art, and preferably, the expression vector of 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.
The host cell may be a prokaryotic cell, 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 one of ordinary skill in the art how to select an appropriate vector and host cell.
gRNA
As used herein, the "gRNA" is also referred to as guide RNA or guide RNA and has a meaning commonly understood by those skilled in the art. In general, the guide RNA may comprise, or consist essentially of, a direct repeat and a guide sequence (guidessequence), also referred to as a spacer (spacer) in the context of an endogenous CRISPR system. grnas may include crRNA and tracrRNA or only crRNA depending on Cas protein on which they depend in different CRISPR systems. The crRNA and tracrRNA may be artificially engineered to fuse to form single guide RNA (sgRNA). In certain instances, the guide sequence is any polynucleotide sequence that is sufficiently complementary to the target sequence (the signature sequence described in the present invention) to hybridize to the target sequence and direct specific binding of the CRISPR/Cas complex to the target sequence, typically having a sequence length of 12-25 nt. The direct repeat sequence can fold to form a specific structure (such as a stem-loop structure) for recognition by the Cas protein to form a complex. The targeting sequence need not be 100% complementary to the signature sequence (target sequence). The targeting sequence is not complementary to the single stranded nucleic acid detector.
In certain embodiments, the degree of complementarity (degree of match) between a 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%, when optimally aligned. 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, the Smith-Waterman algorithm in matlab (Smith-Waterman), Bowtie, Geneius, Biopython, and SeqMan.
The gRNA of the invention can be natural, and can also be artificially modified or designed and synthesized.
Nicking enzyme
Nicking enzymes (also known as Nicking enzymes or Nicking enzymes) are proteins that bind double-stranded DNA and cleave one strand of a double-stranded diploid, cleaving only one strand of the double-stranded DNA to produce a single-stranded nick. Examples of nicking enzymes that can be used to practice the present invention include, but are not limited to, one or any of nb.bbvci, nb.bbci, nb.bsml, nb.bsrdi, nb.bssi, nb.bsti, nt.alwi, nt.bbci, nt.bsmal, nt.bspqi, nt.bstnbl, nt.cvipii, nb.bpuli, or nt.bpuli; more preferably, the nicking enzyme is selected from nb.
Strand-displacing DNA polymerase
Strand displacement DNA polymerase has strand displacement ability, and examples of such DNA polymerases include, but are not limited to, KlenowExo- (New England Biolabs (NEB)), Bst DNA polymerase large fragment (NEB), Vent Exo- (NEB), DeepVent Exo- (NEB), M-MuLV reverse transcriptase (NEB), 9 ℃ NmDNA polymerase (NEB), and Phi29DNA polymerase (NEB). Preferably, the strand displacement DNA polymerase is Klenow Exo-; in other embodiments, the strand displacement DNA polymerase is selected from Klenow Fragment (3 '-5' exo)-) Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, TherminatorTMOne or more of DNA polymerase and Phi29DNA polymerase, preferably Klenow fragment (3 '-5' exo)-)。
Single chain binding proteins
Single-stranded binding protein (SSB or SSBP), also known as Single-stranded DNA binding protein, is a protein responsible for binding to Single-stranded regions of DNA, and can bind to Single-stranded DNA and prevent two complementary strands of Single-stranded DNA from annealing to each other. The genomes of most organisms, including bacteria (e.g., e.coli), viruses (e.g., herpes virus), and mammals, encode at least one SSBP. Suitable examples of single-chain binding proteins include, but are not limited to, e.coli SSB, T4 gp32, T7 gp2.5, RecA, or combinations thereof.
Single-stranded nucleic acid detector
The single-stranded nucleic acid detector of the present invention is a sequence containing 2 to 300 bases, preferably 3 to 200, 4 to 100, 5 to 50, and 5 to 20 bases.
In one embodiment, the single stranded nucleic acid detector may comprise A, T, C, G, U nucleotides; in other embodiments, base modifications may also be included or may be nucleic acid analogs.
The single-stranded nucleic acid detector is used in a detection method or system to report whether a characteristic sequence is contained. The oligonucleotide includes different reporter groups or labeled molecules at both ends, which do not exhibit a reporter signal when in an initial state (i.e., non-cleaved state) and exhibit a detectable signal when the oligonucleotide is cleaved, i.e., exhibits a detectable signal after cleavage and before cleavage. In the present invention, if a detectable signal can be detected, it is reflected that the target nucleic acid contains a characteristic sequence to be detected; alternatively, if the detectable signal is not detectable, it indicates that the target nucleic acid does not contain the signature sequence to be detected.
In one embodiment, the reporter group or the marker molecule comprises a fluorescent group and a quenching group, wherein the fluorescent group is selected from one or any several of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED 460; the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.
In one embodiment, the single stranded nucleic acid detector has a first molecule (e.g., FAM or FITC) attached to the 5 'end and a second molecule (e.g., biotin) attached to the 3' end. The reaction system containing the single-stranded nucleic acid detector is matched with the flow strip to detect the characteristic sequence (preferably, a colloidal gold detection mode). The flow strip is designed with two capture lines, with an antibody that binds to a first molecule (i.e. a first molecular antibody) at the sample contacting end (colloidal gold), an antibody that binds to the first molecular antibody at the first line (control line), and an antibody that binds to a second molecule (i.e. a second molecular antibody, such as avidin) at the second line (test line). As the reaction flows along the strip, the first molecular antibody binds to the first molecule carrying the cleaved or uncleaved oligonucleotide to the capture line, the cleaved reporter will bind to the antibody of the first molecular antibody at the first capture line, and the uncleaved reporter will bind to the second molecular antibody at the second capture line. Binding of the reporter group at each line will result in a strong readout/signal (e.g. color). As more reporters are cut, more signal will accumulate at the first capture line and less signal will appear at the second line. In certain aspects, the invention relates to the use of a flow strip as described herein for detecting nucleic acids. In certain aspects, the invention relates to a method of detecting nucleic acids using a flow strip as defined herein, e.g. a (side) flow test or a (side) flow immunochromatographic assay. In certain aspects, the molecules in the oligonucleotide chain may be substituted for each other, or the position of the molecules may be changed, and the modified forms are also included in the present invention as long as the reporting principle is the same as or similar to that of the present invention.
The detection method can be used for quantitative detection of the characteristic sequence to be detected. The quantitative detection index can be quantified according to the signal intensity of the reporter group, such as the luminous intensity of a fluorescent group, or the width of a color development strip.
Drawings
FIG. 1 shows the effect of adding nicking enzyme, strand displacement polymerase, and single-stranded binding protein to Cas12i detection system sensitivity when detecting PCR reaction products. Wherein line 1 is an experimental group to which nicking enzyme, strand displacement polymerase, and SSB (single-strand binding protein) are added; line 2 is the experimental group to which nicking enzyme and strand displacement polymerase are added; line 3 is the experimental group without nicking enzyme, strand displacement polymerase and SSB; line 4 is a control group to which nicking enzyme, strand displacement polymerase, SSB, but no target nucleic acid, was added; line 5 is a control group to which nicking enzyme, strand displacement polymerase, but no target nucleic acid was added; line 6 is a control group to which no nicking enzyme, strand displacement polymerase, SSB, nor target nucleic acid was added.
FIG. 2 shows the effect of adding nicking enzyme and strand displacement polymerase to the reaction system on the sensitivity of Cas12i detection system when detecting LAMP reaction products. Line 1 is the experimental group with nicking enzyme and strand displacement polymerase added; line 2 is the experimental group without nicking enzyme and strand displacement polymerase; line 3 is a control group to which no target nucleic acid was added.
FIG. 3. Effect of adding nicking enzyme and strand displacing polymerase to the reaction system on the sensitivity of Cas12i detection system when detecting the RPA reaction product. Line 1 is the experimental group with nicking enzyme and strand displacement polymerase added; line 2 is the experimental group without nicking enzyme and strand displacement polymerase; line 3 is a control group to which no target nucleic acid was added.
Detailed description of the preferred embodiments
The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.
The technical scheme is based on the following principle that double-stranded target nucleic acid is obtained in a sample to be detected by an amplification method, the target nucleic acid contains a characteristic sequence, and during actual operation, a proper primer can be designed according to the characteristic sequence to amplify the target nucleic acid by taking the sample to be detected as a template; the primers are provided with recognition sites of the nickase, so that the amplified target nucleic acid containing the enzyme digestion recognition sites of the nickase is guided by the gRNA which can be matched with the characteristic sequence to be recognized by the Cas protein and is combined on the characteristic sequence; subsequently, the Cas protein activates single-stranded nucleic acid cleavage activity, which can cleave a single-stranded nucleic acid detector in the system; in this embodiment, the single-stranded nucleic acid detector has a fluorescent group and a quencher group provided at both ends thereof, respectively, and if the single-stranded nucleic acid detector is cleaved, fluorescence is excited; in other embodiments, both ends of the single-stranded nucleic acid detector may be provided with a label capable of being detected by colloidal gold; in the present invention, a single-stranded nucleic acid can be replaced with a target nucleic acid as a template by adding a nicking enzyme and a strand displacement DNA polymerase; if the target nucleic acid contains the characteristic sequence to be detected, a large amount of single-stranded nucleic acid containing the characteristic sequence to be detected can be obtained by displacement under the action of nicking enzyme and strand displacement DNA polymerase; the Cas protein can recognize a characteristic sequence to be detected in a target nucleic acid, and can also recognize a characteristic sequence to be detected in a replaced single-stranded nucleic acid. Upon recognition of the signature sequence to be detected, the Cas protein is able to cleave the added single-stranded nucleic acid detector and thereby exhibit a detectable signal. The present inventors have found that the sensitivity of the detection can be greatly improved as compared with the control group by adding a nicking enzyme and a strand displacement DNA polymerase.
Chinese patent application (application No. CN202010478129.9, filing date: 2020, 5 months and 29 days) discloses that Cas12i (SEQ ID No.1), Cas12j (SEQ ID No.2), Cas12a (SEQ ID No.3) and Cas12b (SEQ ID No.4) have cleavage activity for different single-stranded nucleic acid detectors (including single-stranded DNA, single-stranded RNA and single-stranded DNA-RNA hybrid), can be used for nucleic acid detection of different samples, and the scheme in the patent application is introduced together in the embodiment.
In the embodiment, the nickase and the strand displacement DNA polymerase are added into a system for detecting the target nucleic acid in the sample by using the Cas protein cutting single-strand nucleic acid detector, and the result shows that the detection sensitivity can be remarkably improved by adding the nickase and the strand displacement DNA polymerase; on the basis, the single-chain binding protein is added, so that the detection sensitivity can be further improved.
Example 1 detection of SARS-CoV-2 Using Cas enzyme
Amplifying orf1ab gene fragment of SARS-CoV-2 by PCR, wherein the PCR primers are designed as follows:
Lamb-i3g2-F:attaccaaggtaaaccggaatttggtgccacttctgc(SEQ ID NO.:5)；
T7-Lamb-j19g2-R:
Figure BDA0002551124740000201
Figure BDA0002551124740000202
(the capitalized base is NbBbvC1 nickase restriction recognition site);
the sequences obtained by PCR amplification are:
attaccaaggtaaacctttggaatttggtgccacttctgctgctcttcaacctgaagaagagcaagaagaagattggttagatgatgatagtcaacaaactgttggtcaacaagacggcagtgaggacaatcagacaactactattcaaacaattgttgaggttcaacctcaattagagatggaacttacaccagttgttcagactattgaagtgaatagtCCTCAGCggattcctatagtgagtcgtatta (SEQ ID NO: 7) (the capitalized base is the NbBbvC1 nickase cleavage recognition site);
the PCR product is used as a target nucleic acid sequence, and the Cas12i protein is used, and the amino acid sequence is as follows: SEQ ID No. 1;
designing gRNA aiming at the target nucleic acid sequence, wherein the sequence is as follows: agagaaugugugcauagucacacggaauuuggugccacuucugc (SEQ ID NO: 8);
a single-stranded nucleic acid detector is used as a Reporter, and the sequence is as follows: 5 '6-FAM-TTTTT-3' BHQ1, and detecting the characteristic sequence by means of fluorescence report.
Furthermore, the accelerating effect of exonuclease on the detection sensitivity of the system was verified by adding dNTPs, nb. bbvcc 1 nickase, klenow strand displacement polymerase or dNTPs, nb. bbvcc 1 nickase, klenow strand displacement polymerase, SSB single strand binding protein T4 gp32 to the system.
In this embodiment, the system has a final Cas12i concentration of 50nM, a final gRNA concentration of 50nM, a final single-stranded nucleic acid detector (Reporter) concentration of 200nM, a nicking enzyme concentration of 0.5U/. mu.l, a strand-displacing polymerase concentration of 0.125U/. mu.l, an SSB concentration of 10ng/ml, and an amount of dNTPs added of 0.8 mM.
As shown in FIG. 1, the addition of nicking enzyme and strand displacement polymerase significantly improves the sensitivity of fluorescence detection and shortens the detection time compared to the absence of nicking enzyme and strand displacement polymerase. The sensitivity of fluorescence detection can be further improved by adding SSB on the basis of adding nickase and strand displacement polymerase.
Example 2 detection of LAMP amplification products Using Cas enzyme
Amplifying orf1ab gene fragment of SARS-CoV-2 by LAMP, wherein LAMP primer is designed as follows:
orf1ab-A-B3 agtctgaacaactggtgtaag(SEQ ID NO.:9)
orf1ab-A-F3 tccagatgaggatgaagaaga(SEQ ID NO.:10)
orf1ab-A-FIP agagcagcagaagtggcacaggtgattgtgaagaagaagag(SEQ ID NO.:11)
orf1ab-A-LB acaaactgttggtcaacaagac(SEQ ID NO.:12)
orf1ab-A-LF ctcatattgagttgatggctca(SEQ ID NO.:13)
orf1ab-A-Nb.BbvCI-BIP
Figure BDA0002551124740000211
recognition sites GCTGAGG of nicking enzyme are added in orf1ab-A-Nb.BbvCI-BIP design, so that the LAMP amplification product contains the recognition sites of Nb.BbvCI nicking enzyme.
The LAMP product was used as a target nucleic acid sequence, and the Cas12i protein, reporter, nicking enzyme, and strand displacement polymerase of example 1 were used to detect a signature sequence by means of fluorescence reporter using gRNA: agagaaugug ugcauaguca cacccaaggu aaaccuuugg aauuugg (SEQ ID NO.: 15).
As shown in fig. 2, when nicking enzymes nb. bbvci and klenow strand displacement polymerase were added to the LAMP reaction product, the fluorescence detection sensitivity was improved and the detection time was shortened as compared with the case where no nicking enzyme and strand displacement polymerase were added.
Example 3 detection of RPA amplification product Using Cas enzyme
Amplifying an N gene segment of SARS-CoV-2 by using RPA, wherein the RPA primer is designed as follows:
N-F:aggcagcagtaggggaacttctcctgctagaat(SEQ ID NO.:16)；
Figure BDA0002551124740000221
wherein the N-DI-R1 was designed by replacing the AA in the original sequence with CA to form the recognition site CATTGC of nicking enzyme Nb. And (3) amplifying fragments:
Figure BDA0002551124740000222
Figure BDA0002551124740000223
(the capitalized base is the cleavage site).
Signature sequences were detected by means of fluorescent Reporter using the RPA product as the target nucleic acid sequence, the Cas12i protein of example 1, and gRNA: agagaaugugugcauaguca cacuugcugc ugcuugacag auu (SEQ ID NO: 19), using a single-stranded nucleic acid detector 5 '-/56-FAM/TTTTT/3 Bio/-3' as the Reporter.
As a result, as shown in fig. 3, when nicking enzymes nb. bsrd1 and klenow strand displacement polymerase were added to the RPA reaction product, the sensitivity of fluorescence detection could be improved and the detection time could be shortened compared to the case where no nicking enzyme and strand displacement polymerase were added.
The above examples 1-3 take Cas12i as an example, and prove that the addition of nicking enzyme, strand displacement DNA polymerase and SSB can improve the sensitivity of the Cas enzyme in detecting target nucleic acid using single-stranded nucleic acid cleavage activity; when other Cas enzymes (such as Cas12i (SEQ ID No.1), Cas12j (SEQ ID No.2), Cas12a (SEQ ID No.3) and Cas12b (SEQ ID No.4) disclosed in Chinese patent application (application No. CN202010478129.9, filing date: 2020, 5 and 29) all have cleavage activity for different single-stranded nucleic acid detectors (including single-stranded DNA, single-stranded RNA and single-stranded DNA-RNA hybrids) and can be used for nucleic acid detection of different samples), the corresponding detection sensitivity can be improved when dNTPs, nickase, strand-displacement DNA polymerase and SSB are added.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Shunheng Biotech Co., Ltd
<120> a method for improving the detection efficiency of a target nucleic acid
<130>P2020-1163
<160>19
<170>PatentIn version 3.5
<210>1
<211>1045
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>1
Met Lys Lys Val Glu Val Ser Arg Pro Tyr Gln Ser Leu Leu Leu Pro
1 5 10 15
Asn His Arg Lys Phe Lys Tyr Leu Asp Glu Thr Trp Asn Ala Tyr Lys
20 25 30
Ser Val Lys Ser Leu Leu His Arg Phe Leu Val Cys Ala Tyr Gly Ala
35 40 45
Val Pro Phe Asn Lys Phe Val Glu Val Val Glu Lys Val Asp Asn Asp
50 55 60
Gln Leu ValLeu Ala Phe Ala Val Arg Leu Phe Arg Leu Val Pro Val
65 70 75 80
Glu Ser Thr Ser Phe Ala Lys Val Asp Lys Ala Asn Leu Ala Lys Ser
85 90 95
Leu Ala Asn His Leu Pro Val Gly Thr Ala Ile Pro Ala Asn Val Gln
100 105 110
Ser Tyr Phe Asp Ser Asn Phe Asp Pro Lys Lys Tyr Met Trp Ile Asp
115 120 125
Cys Ala Trp Glu Ala Asp Arg Leu Ala Arg Glu Met Gly Leu Ser Ala
130 135 140
Ser Gln Phe Ser Glu Tyr Ala Thr Thr Met Leu Trp Glu Asp Trp Leu
145 150 155 160
Pro Leu Asn Lys Asp Asp Val Asn Gly Trp Gly Ser Val Ser Gly Leu
165 170 175
Phe Gly Glu Gly Lys Lys Glu Asp Arg Gln Gln Lys Val Lys Met Leu
180 185 190
Asn Asn Leu Leu Asn Gly Ile Lys Lys Asn Pro Pro Lys Asp Tyr Thr
195 200 205
Gln Tyr Leu Lys Ile Leu Leu Asn Ala Phe Asp Ala Lys Ser His Lys
210 215 220
Glu Ala Val Lys Asn Tyr Lys Gly Asp Ser Thr Gly Arg Thr Ala Ser
225 230 235 240
Tyr Leu Ser Glu Lys Ser Gly Glu Ile Thr Glu Leu Met Leu Glu Gln
245 250 255
Leu Met Ser Asn Ile Gln Arg Asp Ile Gly Asp Lys Gln Lys Glu Ile
260 265 270
Ser Leu Pro Lys Lys Asp Val Val Lys Lys Tyr Leu Glu Ser Glu Ser
275 280 285
Gly Val Pro Tyr Asp Gln Asn Leu Trp Ser Gln Ala Tyr Arg Asn Ala
290 295 300
Ala Ser Ser Ile Lys Lys Thr Asp Thr Arg Asn Phe Asn Ser Thr Leu
305 310 315 320
Glu Lys Phe Lys Asn Glu Val Glu Leu Arg Gly Leu Leu Ser Glu Gly
325 330 335
Asp Asp Val Glu Ile Leu Arg Ser Lys Phe Phe Ser Ser Glu Phe His
340 345 350
Lys Thr Pro Asp Lys Phe Val Ile Lys Pro Glu His Ile Gly Phe Asn
355 360 365
Asn Lys Tyr Asn Val Val Ala Glu Leu Tyr Lys Leu Lys Ala Glu Ala
370 375 380
Thr Asp Phe Glu Ser Ala Phe Ala Thr Val Lys Asp Glu Phe Glu Glu
385 390 395 400
Lys Gly Ile Lys His Pro Ile Lys Asn Ile Leu Glu Tyr Ile Trp Asn
405 410 415
Asn Glu Val Pro Val Glu Lys Trp Gly Arg Val Ala Arg Phe Asn Gln
420 425 430
Ser Glu Glu Lys Leu Leu Arg Ile Lys Ala Asn Pro Thr Val Glu Cys
435 440 445
Asn Gln Gly Met Thr Phe Gly Asn Ser Ala Met Val Gly Glu Val Leu
450 455 460
Arg Ser Asn Tyr Val Ser Lys Lys Gly Ala Leu Val Ser Gly Glu His
465 470 475 480
Gly Gly Arg Leu Ile Gly Gln Asn Asn Met Ile Trp Leu Glu Met Arg
485 490 495
Leu Leu Asn Lys Gly Lys Trp Glu Thr His His Val Pro Thr His Asn
500 505 510
Met Lys Phe Phe Glu Glu Val His Ala Tyr Asn Pro Ser Leu Ala Asp
515 520 525
Ser Val Asn Val Arg Asn Arg Leu Tyr Arg Ser Glu Asp Tyr Thr Gln
530 535 540
Leu Pro Ser Ser Ile Thr Asp Gly Leu Lys Gly Asn Pro Lys Ala Lys
545 550 555 560
Leu Leu Lys Arg Gln His Cys Ala Leu Asn Asn Met Thr Ala Asn Val
565 570 575
Leu Asn Pro Lys Leu Ser Phe Thr Ile Asn Lys Lys Asn Asp Asp Tyr
580 585 590
Thr Val Ile Ile Val His Ser Val Glu Val Ser Lys Pro Arg Arg Glu
595 600 605
Val Leu Val Gly Asp Tyr Leu Val Gly Met Asp Gln Asn Gln Thr Ala
610 615 620
Ser Asn Thr Tyr Ala Val Met Gln Val Val Lys Pro Lys Ser Thr Asp
625 630 635 640
Ala Ile Pro Phe Arg Asn Met Trp Val Arg Phe Val Glu Ser Gly Ser
645 650 655
Ile Glu Ser Arg Thr Leu Asn Ser Arg Gly Glu Tyr Val Asp Gln Leu
660 665 670
Asn His Asp Gly Val Asp Leu Phe Glu Ile Gly Asp Thr Glu Trp Val
675 680 685
Asp Ser Ala Arg Lys Phe Phe Asn Lys Leu Gly Val Lys His Lys Asp
690 695 700
Gly Thr Leu Val Asp Leu Ser Thr Ala Pro Arg Lys Ala Tyr Ala Phe
705 710 715 720
Asn Asn Phe Tyr Phe Lys Thr Met Leu Asn His Leu Arg Ser Asn Glu
725 730 735
Val Asp Leu Thr Leu Leu Arg Asn Glu Ile Leu Arg Val Ala Asn Gly
740 745 750
Arg Phe Ser Pro Met Arg Leu Gly Ser Leu Ser Trp Thr Thr Leu Lys
755 760 765
Ala Leu Gly Ser Phe Lys Ser Leu Val Leu Ser Tyr Phe Asp Arg Leu
770 775 780
Gly Ala Lys Glu Met Val Asp Lys Glu Ala Lys Asp Lys Ser Leu Phe
785 790 795 800
Asp Leu Leu Val Ala Ile Asn Asn Lys Arg Ser Asn Lys Arg Glu Glu
805 810 815
Arg Thr Ser Arg Ile Ala Ser Ser Leu Met Thr Val Ala Gln Lys Tyr
820 825 830
Lys Val Asp Asn Ala Val Val His Val Val Val Glu Gly Asn Leu Ser
835 840 845
Ser Thr Asp Arg Ser Ala Ser Lys Ala His Asn Arg Asn Thr Met Asp
850 855 860
Trp Cys Ser Arg Ala Val Val Lys Lys Leu Glu Asp Met Cys Asn Leu
865 870 875 880
Tyr Gly Phe Asn Ile Lys Gly Val Pro Ala Phe Tyr Thr Ser His Gln
885 890 895
Asp Pro Leu Val His Arg Ala Asp Tyr Asp Asp Pro Lys Pro Ala Leu
900 905 910
Arg Cys Arg Tyr Ser Ser Tyr Ser Arg Ala Asp Phe Ser Lys Trp Gly
915 920 925
Gln Asn Ala Leu Ala Ala Val Val Arg Trp Ala Ser Asn Lys Lys Ser
930 935 940
Asn Thr Cys Tyr Lys Val Gly Ala Val Glu Phe Leu Lys Gln His Gly
945 950 955 960
Leu Phe Ala Asp Lys Lys Leu Thr Val Glu Gln Phe Leu Ser Lys Val
965 970 975
Lys Asp Glu Glu Ile Leu Ile Pro Arg Arg Gly Gly Arg Val Phe Leu
980 985 990
Thr Thr His Arg Leu Leu Ala Glu Ser Thr Phe Val Tyr Leu Asn Gly
995 1000 1005
Val Lys Tyr His Ser Cys Asn Ala Asp Glu Val Ala Ala Val Asn
1010 1015 1020
Ile Cys Leu Asn Asp Trp Val Ile Pro Cys Lys Lys Lys Met Lys
1025 1030 1035
Glu Glu Ser Ser Ala Ser Gly
1040 1045
<210>2
<211>908
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>2
Met Pro Ser Tyr Lys Ser Ser Arg Val Leu Val Arg Asp Val Pro Glu
1 5 10 15
Glu Leu Val Asp His Tyr Glu Arg Ser His Arg Val Ala Ala Phe Phe
20 25 30
Met Arg Leu Leu Leu Ala Met Arg Arg Glu Pro Tyr Ser Leu Arg Met
35 40 45
Arg Asp Gly Thr Glu Arg Glu Val Asp Leu Asp Glu Thr Asp Asp Phe
50 55 60
Leu Arg Ser Ala Gly Cys Glu Glu Pro Asp Ala Val Ser Asp Asp Leu
65 70 75 80
Arg Ser Phe Ala Leu Ala Val Leu His Gln Asp Asn Pro Lys Lys Arg
85 90 95
Ala Phe Leu Glu Ser Glu Asn Cys Val Ser Ile Leu Cys Leu Glu Lys
100 105 110
Ser Ala Ser Gly Thr Arg Tyr Tyr LysArg Pro Gly Tyr Gln Leu Leu
115 120 125
Lys Lys Ala Ile Glu Glu Glu Trp Gly Trp Asp Lys Phe Glu Ala Ser
130 135 140
Leu Leu Asp Glu Arg Thr Gly Glu Val Ala Glu Lys Phe Ala Ala Leu
145 150 155 160
Ser Met Glu Asp Trp Arg Arg Phe Phe Ala Ala Arg Asp Pro Asp Asp
165 170 175
Leu Gly Arg Glu Leu Leu Lys Thr Asp Thr Arg Glu Gly Met Ala Ala
180 185 190
Ala Leu Arg Leu Arg Glu Arg Gly Val Phe Pro Val Ser Val Pro Glu
195 200 205
His Leu Asp Leu Asp Ser Leu Lys Ala Ala Met Ala Ser Ala Ala Glu
210 215 220
Arg Leu Lys Ser Trp Leu Ala Cys Asn Gln Arg Ala Val Asp Glu Lys
225 230 235 240
Ser Glu Leu Arg Lys Arg Phe Glu Glu Ala Leu Asp Gly Val Asp Pro
245 250 255
Glu Lys Tyr Ala Leu Phe Glu Lys Phe Ala Ala Glu Leu Gln Gln Ala
260 265 270
Asp Tyr Asn Val Thr Lys Lys Leu Val Leu AlaVal Ser Ala Lys Phe
275 280 285
Pro Ala Thr Glu Pro Ser Glu Phe Lys Arg Gly Val Glu Ile Leu Lys
290 295 300
Glu Asp Gly Tyr Lys Pro Leu Trp Glu Asp Phe Arg Glu Leu Gly Phe
305 310 315 320
Val Tyr Leu Ala Glu Arg Lys Trp Glu Arg Arg Arg Gly Gly Ala Ala
325 330 335
Val Thr Leu Cys Asp Ala Asp Asp Ser Pro Ile Lys Val Arg Phe Gly
340 345 350
Leu Thr Gly Arg Gly Arg Lys Phe Val Leu Ser Ala Ala Gly Ser Arg
355 360 365
Phe Leu Ile Thr Val Lys Leu Pro Cys Gly Asp Val Gly Leu Thr Ala
370 375 380
Val Pro Ser Arg Tyr Phe Trp Asn Pro Ser Val Gly Arg Thr Thr Ser
385 390 395 400
Asn Ser Phe Arg Ile Glu Phe Thr Lys Arg Thr Thr Glu Asn Arg Arg
405 410 415
Tyr Val Gly Glu Val Lys Glu Ile Gly Leu Val Arg Gln Arg Gly Arg
420 425 430
Tyr Tyr Phe Phe Ile Asp Tyr Asn Phe Asp Pro Glu GluVal Ser Asp
435 440 445
Glu Thr Lys Val Gly Arg Ala Phe Phe Arg Ala Pro Leu Asn Glu Ser
450 455 460
Arg Pro Lys Pro Lys Asp Lys Leu Thr Val Met Gly Ile Asp Leu Gly
465 470 475 480
Ile Asn Pro Ala Phe Ala Phe Ala Val Cys Thr Leu Gly Glu Cys Gln
485 490 495
Asp Gly Ile Arg Ser Pro Val Ala Lys Met Glu Asp Val Ser Phe Asp
500 505 510
Ser Thr Gly Leu Arg Gly Gly Ile Gly Ser Gln Lys Leu His Arg Glu
515 520 525
Met His Asn Leu Ser Asp Arg Cys Phe Tyr Gly Ala Arg Tyr Ile Arg
530 535 540
Leu Ser Lys Lys Leu Arg Asp Arg Gly Ala Leu Asn Asp Ile Glu Ala
545 550 555 560
Arg Leu Leu Glu Glu Lys Tyr Ile Pro Gly Phe Arg Ile Val His Ile
565 570 575
Glu Asp Ala Asp Glu Arg Arg Arg Thr Val Gly Arg Thr Val Lys Glu
580 585 590
Ile Lys Gln Glu Tyr Lys Arg Ile Arg His Gln Phe Tyr Leu ArgTyr
595 600 605
His Thr Ser Lys Arg Asp Arg Thr Glu Leu Ile Ser Ala Glu Tyr Phe
610 615 620
Arg Met Leu Phe Leu Val Lys Asn Leu Arg Asn Leu Leu Lys Ser Trp
625 630 635 640
Asn Arg Tyr His Trp Thr Thr Gly Asp Arg Glu Arg Arg Gly Gly Asn
645 650 655
Pro Asp Glu Leu Lys Ser Tyr Val Arg Tyr Tyr Asn Asn Leu Arg Met
660 665 670
Asp Thr Leu Lys Lys Leu Thr Cys Ala Ile Val Arg Thr Ala Lys Glu
675 680 685
His Gly Ala Thr Leu Val Ala Met Glu Asn Ile Gln Arg Val Asp Arg
690 695 700
Asp Asp Glu Val Lys Arg Arg Lys Glu Asn Ser Leu Leu Ser Leu Trp
705 710 715 720
Ala Pro Gly Met Val Leu Glu Arg Val Glu Gln Glu Leu Lys Asn Glu
725 730 735
Gly Ile Leu Ala Trp Glu Val Asp Pro Arg His Thr Ser Gln Thr Ser
740 745 750
Cys Ile Thr Asp Glu Phe Gly Tyr Arg Ser Leu Val Ala Lys Asp Thr
755 760 765
Phe Tyr Phe Glu Gln Asp Arg Lys Ile His Arg Ile Asp Ala Asp Val
770 775 780
Asn Ala Ala Ile Asn Ile Ala Arg Arg Phe Leu Thr Arg Tyr Arg Ser
785 790 795 800
Leu Thr Gln Leu Trp Ala Ser Leu Leu Asp Asp Gly Arg Tyr Leu Val
805 810 815
Asn Val Thr Arg Gln His Glu Arg Ala Tyr Leu Glu Leu Gln Thr Gly
820 825 830
Ala Pro Ala Ala Thr Leu Asn Pro Thr Ala Glu Ala Ser Tyr Glu Leu
835 840 845
Val Gly Leu Ser Pro Glu Glu Glu Glu Leu Ala Gln Thr Arg Ile Lys
850 855 860
Arg Lys Lys Arg Glu Pro Phe Tyr Arg His Glu Gly Val Trp Leu Thr
865 870 875 880
Arg Glu Lys His Arg Glu Gln Val His Glu Leu Arg Asn Gln Val Leu
885 890 895
Ala Leu Gly Asn Ala Lys Ile Pro Glu Ile Arg Thr
900 905
<210>3
<211>1228
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>3
Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr
1 5 10 15
Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp
20 25 30
Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys
35 40 45
Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp
50 55 60
Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu
65 70 75 80
Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn
85 90 95
Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn
100 105 110
Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu
115 120 125
Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe
130 135 140
Asn Gly Phe Thr ThrAla Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn
145 150 155 160
Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile
165 170 175
Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys
180 185 190
Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys
195 200 205
Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe
210 215 220
Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile
225 230 235 240
Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn
245 250 255
Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys
260 265 270
Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser
275 280 285
Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe
290 295 300
Arg Asn Thr Leu Asn Lys AsnSer Glu Ile Phe Ser Ser Ile Lys Lys
305 310 315 320
Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile
325 330 335
Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe
340 345 350
Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp
355 360 365
Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp
370 375 380
Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu
385 390 395 400
Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu
405 410 415
Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser
420 425 430
Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys
435 440 445
Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys
450 455 460
Ser Phe Glu Asn Tyr Ile Lys Ala PhePhe Gly Glu Gly Lys Glu Thr
465 470 475 480
Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile
485 490 495
Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr
500 505 510
Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro
515 520 525
Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala
530 535 540
Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys
545 550 555 560
Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly
565 570 575
Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met
580 585 590
Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro
595 600 605
Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly
610 615 620
Asp Met Phe Asn Leu Asn Asp Cys His Lys LeuIle Asp Phe Phe Lys
625 630 635 640
Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn
645 650 655
Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu
660 665 670
Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys
675 680 685
Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile
690 695 700
Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His
705 710 715 720
Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile
725 730 735
Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys
740 745 750
Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys
755 760 765
Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr
770 775 780
Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu HisIle Pro Ile
785 790 795 800
Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val
805 810 815
Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp
820 825 830
Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly
835 840 845
Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn
850 855 860
Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu
865 870 875 880
Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile
885 890 895
Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys
900 905 910
Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn
915 920 925
Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln
930 935 940
Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val AspLys
945 950 955 960
Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile
965 970 975
Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe
980 985 990
Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr
995 1000 1005
Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp
1010 1015 1020
Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro
1025 1030 1035
Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser
1040 1045 1050
Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr
1055 1060 1065
Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val
1070 1075 1080
Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu
1085 1090 1095
Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala
11001105 1110
Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met
1115 1120 1125
Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly
1130 1135 1140
Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp
1145 1150 1155
Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala
1160 1165 1170
Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala
1175 1180 1185
Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp
1190 1195 1200
Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp
1205 1210 1215
Leu Glu Tyr Ala Gln Thr Ser Val Lys His
1220 1225
<210>4
<211>1129
<212>PRT
<213> Artificial sequence (artificial sequence)
<400>4
Met Ala Val Lys Ser Ile Lys Val Lys Leu Arg Leu Asp Asp Met Pro
1 5 1015
Glu Ile Arg Ala Gly Leu Trp Lys Leu His Lys Glu Val Asn Ala Gly
20 25 30
Val Arg Tyr Tyr Thr Glu Trp Leu Ser Leu Leu Arg Gln Glu Asn Leu
35 40 45
Tyr Arg Arg Ser Pro Asn Gly Asp Gly Glu Gln Glu Cys Asp Lys Thr
50 55 60
Ala Glu Glu Cys Lys Ala Glu Leu Leu Glu Arg Leu Arg Ala Arg Gln
65 70 75 80
Val Glu Asn Gly His Arg Gly Pro Ala Gly Ser Asp Asp Glu Leu Leu
85 90 95
Gln Leu Ala Arg Gln Leu Tyr Glu Leu Leu Val Pro Gln Ala Ile Gly
100 105 110
Ala Lys Gly Asp Ala Gln Gln Ile Ala Arg Lys Phe Leu Ser Pro Leu
115 120 125
Ala Asp Lys Asp Ala Val Gly Gly Leu Gly Ile Ala Lys Ala Gly Asn
130 135 140
Lys Pro Arg Trp Val Arg Met Arg Glu Ala Gly Glu Pro Gly Trp Glu
145 150 155 160
Glu Glu Lys Glu Lys Ala Glu Thr Arg Lys Ser Ala Asp Arg Thr Ala
165 170 175
Asp Val Leu Arg Ala Leu Ala Asp Phe Gly Leu Lys Pro Leu Met Arg
180 185 190
Val Tyr Thr Asp Ser Glu Met Ser Ser Val Glu Trp Lys Pro Leu Arg
195 200 205
Lys Gly Gln Ala Val Arg Thr Trp Asp Arg Asp Met Phe Gln Gln Ala
210 215 220
Ile Glu Arg Met Met Ser Trp Glu Ser Trp Asn Gln Arg Val Gly Gln
225 230 235 240
Glu Tyr Ala Lys Leu Val Glu Gln Lys Asn Arg Phe Glu Gln Lys Asn
245 250 255
Phe Val Gly Gln Glu His Leu Val His Leu Val Asn Gln Leu Gln Gln
260 265 270
Asp Met Lys Glu Ala Ser Pro Gly Leu Glu Ser Lys Glu Gln Thr Ala
275 280 285
His Tyr Val Thr Gly Arg Ala Leu Arg Gly Ser Asp Lys Val Phe Glu
290 295 300
Lys Trp Gly Lys Leu Ala Pro Asp Ala Pro Phe Asp Leu Tyr Asp Ala
305 310 315 320
Glu Ile Lys Asn Val Gln Arg Arg Asn Thr Arg Arg Phe Gly Ser His
325 330 335
Asp Leu Phe Ala Lys Leu Ala Glu Pro Glu Tyr Gln Ala Leu Trp Arg
340 345 350
Glu Asp Ala Ser Phe Leu Thr Arg Tyr Ala Val Tyr Asn Ser Ile Leu
355 360 365
Arg Lys Leu Asn His Ala Lys Met Phe Ala Thr Phe Thr Leu Pro Asp
370 375 380
Ala Thr Ala His Pro Ile Trp Thr Arg Phe Asp Lys Leu Gly Gly Asn
385 390 395 400
Leu His Gln Tyr Thr Phe Leu Phe Asn Glu Phe Gly Glu Arg Arg His
405 410 415
Ala Ile Arg Phe His Lys Leu Leu Lys Val Glu Asn Gly Val Ala Arg
420 425 430
Glu Val Asp Asp Val Thr Val Pro Ile Ser Met Ser Glu Gln Leu Asp
435 440 445
Asn Leu Leu Pro Arg Asp Pro Asn Glu Pro Ile Ala Leu Tyr Phe Arg
450 455 460
Asp Tyr Gly Ala Glu Gln His Phe Thr Gly Glu Phe Gly Gly Ala Lys
465 470 475 480
Ile Gln Cys Arg Arg Asp Gln Leu Ala His Met His Arg Arg Arg Gly
485 490 495
Ala Arg Asp Val Tyr Leu Asn Val Ser Val Arg Val Gln Ser Gln Ser
500 505 510
Glu Ala Arg Gly Glu Arg Arg Pro Pro Tyr Ala Ala Val Phe Arg Leu
515 520 525
Val Gly Asp Asn His Arg Ala Phe Val His Phe Asp Lys Leu Ser Asp
530 535 540
Tyr Leu Ala Glu His Pro Asp Asp Gly Lys Leu Gly Ser Glu Gly Leu
545 550 555 560
Leu Ser Gly Leu Arg Val Met Ser Val Asp Leu Gly Leu Arg Thr Ser
565 570 575
Ala Ser Ile Ser Val Phe Arg Val Ala Arg Lys Asp Glu Leu Lys Pro
580 585 590
Asn Ser Lys Gly Arg Val Pro Phe Phe Phe Pro Ile Lys Gly Asn Asp
595 600 605
Asn Leu Val Ala Val His Glu Arg Ser Gln Leu Leu Lys Leu Pro Gly
610 615 620
Glu Thr Glu Ser Lys Asp Leu Arg Ala Ile Arg Glu Glu Arg Gln Arg
625 630 635 640
Thr Leu Arg Gln Leu Arg Thr Gln Leu Ala Tyr Leu Arg Leu Leu Val
645 650 655
Arg Cys Gly Ser Glu Asp Val Gly Arg Arg Glu Arg Ser Trp Ala Lys
660 665 670
Leu Ile Glu Gln Pro Val Asp Ala Ala Asn His Met Thr Pro Asp Trp
675 680 685
Arg Glu Ala Phe Glu Asn Glu Leu Gln Lys Leu Lys Ser Leu His Gly
690 695 700
Ile Cys Ser Asp Lys Glu Trp Met Asp Ala Val Tyr Glu Ser Val Arg
705 710 715 720
Arg Val Trp Arg His Met Gly Lys Gln Val Arg Asp Trp Arg Lys Asp
725 730 735
Val Arg Ser Gly Glu Arg Pro Lys Ile Arg Gly Tyr Ala Lys Asp Val
740 745 750
Val Gly Gly Asn Ser Ile Glu Gln Ile Glu Tyr Leu Glu Arg Gln Tyr
755 760 765
Lys Phe Leu Lys Ser Trp Ser Phe Phe Gly Lys Val Ser Gly Gln Val
770 775 780
Ile Arg Ala Glu Lys Gly Ser Arg Phe Ala Ile Thr Leu Arg Glu His
785 790 795 800
Ile Asp His Ala Lys Glu Asp Arg Leu Lys Lys Leu Ala Asp Arg Ile
805 810 815
Ile Met Glu Ala Leu Gly Tyr Val Tyr Ala Leu Asp Glu Arg Gly Lys
820 825 830
Gly Lys Trp Val Ala Lys Tyr Pro Pro Cys Gln Leu Ile Leu Leu Glu
835 840 845
Glu Leu Ser Glu Tyr Gln Phe Asn Asn Asp Arg Pro Pro Ser Glu Asn
850 855 860
Asn Gln Leu Met Gln Trp Ser His Arg Gly Val Phe Gln Glu Leu Ile
865 870 875 880
Asn Gln Ala Gln Val His Asp Leu Leu Val Gly Thr Met Tyr Ala Ala
885 890 895
Phe Ser Ser Arg Phe Asp Ala Arg Thr Gly Ala Pro Gly Ile Arg Cys
900 905 910
Arg Arg Val Pro Ala Arg Cys Thr Gln Glu His Asn Pro Glu Pro Phe
915 920 925
Pro Trp Trp Leu Asn Lys Phe Val Val Glu His Thr Leu Asp Ala Cys
930 935 940
Pro Leu Arg Ala Asp Asp Leu Ile Pro Thr Gly Glu Gly Glu Ile Phe
945 950 955 960
Val Ser Pro Phe Ser Ala Glu Glu Gly Asp Phe His Gln Ile His Ala
965 970 975
Asp Leu Asn Ala Ala Gln Asn Leu Gln Gln Arg Leu Trp Ser Asp Phe
980 985 990
Asp Ile Ser Gln Ile Arg Leu Arg Cys Asp Trp Gly Glu Val Asp Gly
995 1000 1005
Glu Leu Val Leu Ile Pro Arg Leu Thr Gly Lys Arg Thr Ala Asp
1010 1015 1020
Ser Tyr Ser Asn Lys Val Phe Tyr Thr Asn Thr Gly Val Thr Tyr
1025 1030 1035
Tyr Glu Arg Glu Arg Gly Lys Lys Arg Arg Lys Val Phe Ala Gln
1040 1045 1050
Glu Lys Leu Ser Glu Glu Glu Ala Glu Leu Leu Val Glu Ala Asp
1055 1060 1065
Glu Ala Arg Glu Lys Ser Val Val Leu Met Arg Asp Pro Ser Gly
1070 1075 1080
Ile Ile Asn Arg Gly Asn Trp Thr Arg Gln Lys Glu Phe Trp Ser
1085 1090 1095
Met Val Asn Gln Arg Ile Glu Gly Tyr Leu Val Lys Gln Ile Arg
1100 1105 1110
Ser Arg Val Pro Leu Gln Asp Ser Ala Cys Glu Asn Thr Gly Asp
1115 1120 1125
Ile
<210>5
<211>37
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>5
attaccaagg taaaccggaa tttggtgcca cttctgc 37
<210>6
<211>58
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>6
taatacgact cactatagga atccgctgag gactattcac ttcaatagtc tgaacaac 58
<210>7
<211>252
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>7
attaccaagg taaacctttg gaatttggtg ccacttctgc tgctcttcaa cctgaagaag 60
agcaagaaga agattggtta gatgatgata gtcaacaaac tgttggtcaa caagacggca 120
gtgaggacaa tcagacaact actattcaaa caattgttga ggttcaacct caattagaga 180
tggaacttac accagttgtt cagactattg aagtgaatag tcctcagcgg attcctatag 240
tgagtcgtat ta 252
<210>8
<211>44
<212>RNA
<213> Artificial sequence (artificial sequence)
<400>8
agagaaugug ugcauaguca cacggaauuu ggugccacuu cugc 44
<210>9
<211>21
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>9
agtctgaaca actggtgtaa g 21
<210>10
<211>21
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>10
tccagatgag gatgaagaag a 21
<210>11
<211>41
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>11
agagcagcag aagtggcaca ggtgattgtg aagaagaaga g 41
<210>12
<211>22
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>12
acaaactgtt ggtcaacaag ac 22
<210>13
<211>22
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>13
ctcatattga gttgatggct ca 22
<210>14
<211>47
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>14
tcaacctgaa gaagagcaag aagctgaggc tgattgtcct cactgcc 47
<210>15
<211>47
<212>RNA
<213> Artificial sequence (artificial sequence)
<400>15
agagaaugug ugcauaguca cacccaaggu aaaccuuugg aauuugg 47
<210>16
<211>33
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>16
aggcagcagt aggggaactt ctcctgctag aat 33
<210>17
<211>38
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>17
gttggccttt accagacaca ttgctctcaa gctggttc 38
<210>18
<211>122
<212>DNA
<213> Artificial sequence (artificial sequence)
<400>18
aggcagcagt aggggaactt ctcctgctag aatggctggc aatggcggtg atgctgctct 60
tgctttgctg ctgcttgaca gattgaacca gcttgagagc aaaatgtctg gtaaaggcca 120
ac 122
<210>19
<211>43
<212>RNA
<213> Artificial sequence (artificial sequence)
<400>19
agagaaugug ugcauaguca cacuugcugc ugcuugacag auu 43

Claims (10)

1. A method for detecting the presence of a signature sequence to be detected in a target nucleic acid based on CRISPR technology, the method comprising:
(1) providing target nucleic acid, nickase, strand displacement DNA polymerase, dNTPs, gRNA, Cas protein and a single-stranded nucleic acid detector, wherein the target nucleic acid is provided with an enzyme cutting recognition site of the nickase;
(2) the nickase cuts the enzyme cutting recognition site to form a gap, and the strand displacement DNA polymerase extends from the gap to displace at least one single-stranded nucleic acid;
(3) the gRNA can target a characteristic sequence to be detected, the Cas protein can recognize the characteristic sequence to be detected under the action of the gRNA, and the Cas protein can cut the single-stranded nucleic acid detector;
(4) the single-stranded nucleic acid detector exhibits a detectable signal after cleavage by the Cas protein compared to the single-stranded nucleic acid detector before cleavage by the Cas protein;
(5) testing whether the detectable signal of step (4) can be detected; if the detectable signal in the step (4) can be detected, reflecting that the target nucleic acid contains the characteristic sequence to be detected; alternatively, if the detectable signal in step (4) is not detected, it is reflected that the target nucleic acid does not contain the signature sequence to be detected.
2. The method of claim 1, wherein the Cas protein is a protein with trans cleavage activity, preferably, a type V CRISPR/Cas effector protein; more preferably, the V-type Cas protein is selected from any one or any combination of Cas12 and Cas14 family proteins;
preferably, the Cas12 family protein is selected from one or any combination of Cas12i, Cas12j, Cas12a, Cas12b, Cas12d, Cas12e, Cas12f, Cas12g and Cas12 h;
more preferably, the Cas12 family protein is one or a combination of any two of Cas12i, Cas12j, Cas12a and Cas12 b.
3. The method of claim 1 or 2, wherein the detectable signal is detected by: vision-based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization, colloidal phase transition/dispersion, electrochemical detection, and semiconductor-based detection.
4. The method of any one of claims 1-3, further comprising providing a single-chain binding protein (SSB).
5. The method according to any one of claims 1 to 4, wherein the nicking enzyme is a tool enzyme capable of recognizing double strands of DNA but cleaving only single strands where the recognition site is located, preferably the nicking enzyme is selected from one or more of Nb.BbCI, Nb.Bsml, Nb.BsrDI, Nb.BssSI, NbBstI, Nt.AlwI, Nt.BbCI, Nt.BsmAl, Nt.BspQl or Nt.BstNBl; more preferably, the nicking enzyme is selected from nb.
6. The method of any one of claims 1 to 5, wherein the strand-displacing DNA polymerase is selected from Klenow Fragment (3 '-5' exo)-) Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, TherminatorTMOne or more of DNA polymerase and Phi29DNA polymerase, preferably Klenow fragment (3 '-5' exo)-)。
7. The method of any one of claims 1 to 6,
the 5 'end and the 3' end of the single-stranded nucleic acid detector are respectively provided with different reporter groups, when the single-stranded nucleic acid detector is cut, a detectable reporter signal can be shown, and whether the target nucleic acid contains a characteristic sequence to be detected is reflected by the existence of the reporter signal;
or, different labeled molecules are respectively arranged at the 5 'end and the 3' end of the single-stranded nucleic acid detector, and the results of the single-stranded nucleic acid detector before and after cleavage by the Cas protein are detected by a lateral flow test strip, for example, a colloidal gold test strip, so as to reflect whether the target nucleic acid contains the characteristic sequence to be detected.
8. A system for detecting the presence of a signature sequence to be detected in a target nucleic acid based on CRISPR technology, the system comprising: the kit comprises nickase, strand displacement DNA polymerase, dNTPs, gRNA, Cas protein and a single-stranded nucleic acid detector, wherein the target nucleic acid is provided with a restriction enzyme cutting recognition site of the nickase; preferably, the system further comprises a single chain binding protein.
9. A kit for detecting the presence of a signature sequence to be detected in a target nucleic acid based on CRISPR technology, comprising the system of claim 7; preferably, the kit further comprises a primer for amplifying the target nucleic acid; more preferably, the primer is provided with an enzyme cutting recognition site of the nicking enzyme.
10. Use of the system according to claim 8 or the kit according to claim 9 for diagnosing or detecting the presence of a characteristic sequence to be detected in a sample to be tested; preferably, the first and second liquid crystal materials are,
the characteristic sequence to be detected is a virus specific sequence, a bacterium specific sequence, a characteristic sequence related to diseases, a specific mutation site or an SNP site; more preferably, the virus is a plant virus or an animal virus; more preferably, the virus is a coronavirus; more preferably, the target nucleic acid is derived from a virus, bacterium, microorganism, soil, water source, human, non-human animal or plant.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112831544A (en) * 2020-12-31 2021-05-25 华南农业大学 Biological detection method and biological detection device based on CRISPR/Cas12a system
WO2021238128A1 (en) * 2020-05-28 2021-12-02 上海科技大学 Genome editing system and method
CN113913498A (en) * 2020-12-14 2022-01-11 山东舜丰生物科技有限公司 Method for detecting target mutation based on CRISPR technology
CN113913499A (en) * 2020-12-25 2022-01-11 山东舜丰生物科技有限公司 Method for detecting target mutation by using Cas12j effector protein
CN114134218A (en) * 2021-12-02 2022-03-04 中国疾病预防控制中心传染病预防控制所 Fluorescent detection method based on CRISPR-Cas12a
WO2022100527A1 (en) * 2020-11-11 2022-05-19 山东舜丰生物科技有限公司 Novel cas enzyme and system and use thereof
WO2024006552A1 (en) * 2022-07-01 2024-01-04 Sherlock Biosciences, Inc. Ambient temperature nucleic acid amplification and detection

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140309142A1 (en) * 2012-04-16 2014-10-16 Jingdong Tian Method of on-chip nucleic acid molecule synthesis
CN110551800A (en) * 2018-06-03 2019-12-10 上海吐露港生物科技有限公司 Application of high-temperature-resistant Cas protein, and detection method and kit of target nucleic acid molecule
WO2020006067A1 (en) * 2018-06-26 2020-01-02 The Broad Institute, Inc. Crispr double nickase based amplification compositions, systems, and methods
US20200063126A1 (en) * 2018-03-14 2020-02-27 Arbor Biotechnologies, Inc. Novel crispr dna targeting enzymes and systems
CN111094588A (en) * 2017-07-14 2020-05-01 上海吐露港生物科技有限公司 Application of Cas protein, and detection method and kit of target nucleic acid molecule

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140309142A1 (en) * 2012-04-16 2014-10-16 Jingdong Tian Method of on-chip nucleic acid molecule synthesis
CN111094588A (en) * 2017-07-14 2020-05-01 上海吐露港生物科技有限公司 Application of Cas protein, and detection method and kit of target nucleic acid molecule
US20200063126A1 (en) * 2018-03-14 2020-02-27 Arbor Biotechnologies, Inc. Novel crispr dna targeting enzymes and systems
CN110551800A (en) * 2018-06-03 2019-12-10 上海吐露港生物科技有限公司 Application of high-temperature-resistant Cas protein, and detection method and kit of target nucleic acid molecule
WO2020006067A1 (en) * 2018-06-26 2020-01-02 The Broad Institute, Inc. Crispr double nickase based amplification compositions, systems, and methods

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WINSTON X. YAN等: "Functionally diverse type V CRISPR-Cas systems", 《SCIENCE》 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021238128A1 (en) * 2020-05-28 2021-12-02 上海科技大学 Genome editing system and method
WO2022100527A1 (en) * 2020-11-11 2022-05-19 山东舜丰生物科技有限公司 Novel cas enzyme and system and use thereof
CN113913498A (en) * 2020-12-14 2022-01-11 山东舜丰生物科技有限公司 Method for detecting target mutation based on CRISPR technology
CN113913499A (en) * 2020-12-25 2022-01-11 山东舜丰生物科技有限公司 Method for detecting target mutation by using Cas12j effector protein
CN112831544A (en) * 2020-12-31 2021-05-25 华南农业大学 Biological detection method and biological detection device based on CRISPR/Cas12a system
CN112831544B (en) * 2020-12-31 2024-06-14 华南农业大学 Biological detection method and biological detection device based on CRISPR/Cas12a system
CN114134218A (en) * 2021-12-02 2022-03-04 中国疾病预防控制中心传染病预防控制所 Fluorescent detection method based on CRISPR-Cas12a
CN114134218B (en) * 2021-12-02 2022-10-28 中国疾病预防控制中心传染病预防控制所 Fluorescent detection method based on CRISPR-Cas12a
WO2024006552A1 (en) * 2022-07-01 2024-01-04 Sherlock Biosciences, Inc. Ambient temperature nucleic acid amplification and detection

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