WO2020259210A1 - 一种检测非洲猪瘟病毒的方法和试剂盒 - Google Patents

一种检测非洲猪瘟病毒的方法和试剂盒 Download PDF

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WO2020259210A1
WO2020259210A1 PCT/CN2020/093456 CN2020093456W WO2020259210A1 WO 2020259210 A1 WO2020259210 A1 WO 2020259210A1 CN 2020093456 W CN2020093456 W CN 2020093456W WO 2020259210 A1 WO2020259210 A1 WO 2020259210A1
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detection system
fever virus
swine fever
african swine
detection
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PCT/CN2020/093456
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French (fr)
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吴尧
杨海平
谢云鹤
李秋实
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苏州克睿基因生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • the invention belongs to the field of virus detection, and particularly relates to a system for detecting African swine fever virus, a detection method and a kit.
  • the invention can be used in the detection of African swine fever virus or other viruses that can infect pigs.
  • African swine fever is an acute, highly infectious disease with clinical symptoms such as hemorrhagic fever caused by the African swine fever virus (ASFV), with a fatality rate higher than 90%.
  • ASFV is a linear double-stranded DNA virus with a genome length of about 170 kb to 193 kb, which can encode 150 to 200 proteins.
  • ASFV is mainly transmitted cyclically among wild boars, domestic pigs and soft ticks. Due to its extremely high infectivity and lethality, it has caused a serious economic burden on the pig industry in the affected areas.
  • ASF is listed as one of the statutory reported animal diseases by the World Health Organization (OIE), and is listed as a class of animal infectious diseases in China.
  • ASF was first discovered in Africa in the 1960s, and then spread to Europe, the Americas and other continents. More than 60 different ASFV strains have been found. According to the P72 gene, it can be divided into 24 genotypes. In August 2018, the first ASF epidemic was confirmed in Shenyang, my country. About 400 infected pigs died within one month after the outbreak. Subsequently, many provinces and regions in my country have successively discovered ASF epidemics, and the control of the ASF epidemic is urgent.
  • Antigen-antibody-based immunoassays include direct or indirect fluorescent antibody detection and enzyme-linked immunosorbent assay detection. Although the process is simple, the sensitivity is not enough, and false negatives and missed detection are prone to occur.
  • the PCR method has certain sensitivity and specificity, and is a more popular detection method on the market, including the routine PCR and fluorescent real-time quantitative PCR method recommended by OIE for routine laboratory diagnosis (Agüero M, et al. J Clin Microbiol 2003, 41 :4431–4434;). However, some research reports have shown that nucleotide mismatches between primers and viral target genes in the method recommended by the OIE will reduce the sensitivity and specificity of PCR (Gallardo C et al. J Clin Microbiol.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the CRISPR-Cas system is an RNA-guided adaptive immune system in microorganisms. When viruses invade bacteria, the bacteria can capture fragments of foreign genetic material and integrate them into CRISPR sequences in their own genomes.
  • the CRISPR RNA (crRNA) generated by the transcription of the CRISPR sequence can be combined with the CRISPR binding protein (Cas nuclease), and provides binding and cleavage specificity for the Cas nuclease through base pairing with the target nucleic acid sequence (Cong et al.
  • Cas9 nuclease is the first Cas protein widely recognized and used in genome editing, but scientists have now expanded the scope of Cas protein to more other types of Cas proteins, such as the newly reported Cas13a, Cas12a and Cas14a proteins Wait.
  • Cas13a nuclease cleaves specific target RNA under the action of crRNA, and at the same time activates its own nuclease activity to cut non-specific RNA.
  • Cas12a and other V-type CRISPR-Cas12 enzymes have the characteristics of target-activated non-specific ssDNase cleavage.
  • the DETECTR nucleic acid detection method was created by combining Cas12a with isothermal amplification, which achieved the highest level of DNA detection.
  • the ultra-high sensitivity can quickly and specifically detect human papillomavirus in patient samples (Doudna JA et al. Science. 2018 Apr 27; 360(6387):436-439).
  • the purpose of the present invention is to provide a rapid nucleic acid detection system and detection method for African swine fever virus based on Cas12 protein.
  • a nucleic acid detection system for detecting African swine fever virus including:
  • the Cas12a protein is selected from FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a.
  • the detection system of technical solution 4 wherein the Cas12a protein has the sequence shown in SEQ ID No: 1; or has a sequence that is at least 80% identical to the sequence shown in SEQ ID No: 1; or is the same as SEQ ID No: 1
  • the sequence shown has one or several amino acid substitutions, deletions and insertions of Cas12a protein variants.
  • the gene fragment of African swine fever virus for example, the K205R gene fragment has a length of at least 15 bp, preferably at least 20 bp.
  • the detection system described in any one of the foregoing technical solutions further comprises a nucleic acid amplification system of African swine fever virus.
  • amplification system is a recombinase polymerase amplification (RPA) system.
  • RPA recombinase polymerase amplification
  • nucleic acid of the African swine fever virus is derived from swine tissue, which is preferably whole blood, swine plasma, swine serum, swine urine, swine saliva or Porcine oral mucosa.
  • a nucleic acid detection method for detecting African swine fever virus comprising:
  • the nucleic acid sample is sample DNA extracted from African swine fever virus, or target DNA obtained by amplification of sample DNA extracted from African swine fever virus.
  • nucleic acid sample is from pig tissue, which is preferably whole blood, pig plasma, pig serum, pig urine, pig saliva or pig oral mucosa.
  • the method for rapid detection of African swine fever virus based on gene editing enzymes includes a basic detection system and a post-amplification detection system. Compared with the prior art, it has one or more of the following advantages:
  • the application of the present invention can realize the detection of aM-level DNA with a detection limit as low as 10 copies. In addition to the rapid qualitative detection of ASFV, it can also realize the early screening of ASFV. The missed detection rate due to false negatives is extremely low ;
  • the present invention can complete the detection in as little as half an hour;
  • the application of the invention can realize rapid detection of DNA viruses such as ASFV.
  • Figure 1 Schematic diagram of the results of 2% agarose gel electrophoresis (with T7 and without T7) of the product of the RPA reaction using synthetic K205R gene plasmid DNA as a template provided by the embodiment of the present invention
  • Figure 2 Schematic diagram of fluorescence detection results of specific cleavage of K205R gene and non-specific cleavage of ssDNA fluorescent probe after T1-crRNA1 and T2-crRNA1 form a complex with Cas12a protein provided by the embodiment of the present invention
  • Figure 3 The embodiment of the present invention provides a schematic diagram of the fluorescence detection results of Cas12a basic detection of K205R genes with different initial amounts;
  • Figure 4 Sensitivity schematic diagram of the African swine fever virus detection method based on Cas12a and RPA provided by the embodiment of the present invention (consecutive time and certain time);
  • Figure 5 Schematic diagram of the combined detection sensitivity of Cas12a and RPA for another gene target.
  • CRISPR refers to clustered regularly spaced short palindromic repeats (clustered regularly interspaced short palindromic repeats), which is the immune system of many prokaryotes.
  • Cas protein refers to CRISPR/Cas effector protein.
  • Current research has found that there are two main types of CRISPR-Cas systems.
  • the first type is Class1 containing multi-subunit protein effector complexes and Class2 single-subunit protein effector complexes.
  • the Class1 CRISPR-Cas system is the most common in bacteria and archaea (including all hyperthermophiles). This type of protein accounts for about 90% of all identified CRISPR-Cas proteins (Makarova KS, et al. An updated evolutionary). classification of CRISPR-Cas systems.Nat.Rev.Microbiol.2015;13:722–736).
  • Class 2 The CRISPR-Cas system is almost exclusively found in bacteria.
  • the Cas proteins available for this system mainly include type II, V, and VI effector proteins, accounting for about 10% of the CRISPR-Cas protein, mainly including the commonly used Cas9 protein (type II). ), and the newly discovered Cas12 (V type), Cas13 (VI) type and Cas14 (V type) proteins (Chylinski K et al. Nucleic Acids Res. 2014; 42:6091-6105; Shmakov S, et al. Mol.Cell.2015; 60: 385–397; Sergey Shmakov et al. Nat Rev Microbiol. 2017 March; 15(3): 169–182; Doudna JAet al. Science. 2018 Nov 16; 362(6416): 839-842 ). Due to the relatively simple effect complex composition of the Class 2 CRISPR-Cas system, it has become one of the hottest gene editing tools for scientific research.
  • CRISPR/Cas system and “CRISPR system” have the same meaning.
  • the Cas protein involved in the technical scheme of the present invention is the Cas12 (V-type CRISPR/Cas effector protein) protein family.
  • the Cas12 family includes Cas12a, Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h and Cas12i, etc.; for the definition of Cas12, please refer to the document Koonin et al., CurrOpinMicrobiol. 2017 June; 37:67 -78: "Diversity, classification and evolution of CRISPR-Cas systems", the content of this document is part of this patent application.
  • These Cas proteins can be derived from different genera, and their enzyme activities may also be different.
  • the Cas12 protein of the present invention is a Cas12a protein.
  • Cas12a protein (formerly known as "Cpf1") refers to a crRNA-dependent endonuclease, which is a type V enzyme in the CRISPR system classification.
  • the Cas12a protein of the present invention may be Cas12a proteins derived from different species, such as one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, and Lb4Cas12a.
  • the Cas12a protein is preferably LbCas12a; more preferably Cas12a (SEQIDNo:1) of the sequence shown in Figure 5, or at least 80%, 83%, 85%, 86%, 87%, 88%, 89% with SEQIDNo:1. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical Cas12a, or have one or more of SEQIDNo:1
  • one or several amino acid deletions, substitutions or additions of Cas12a variants refer to 1, 2, 3, 4, 5, 6, 7, 8, 9 Or deletion, substitution or addition of 10 amino acids.
  • Cas12 proteins such as Cas12a, Cas12b, Cas12c, Cas12d, and Cas12e, are fusion proteins.
  • the heterologous polypeptide provides subcellular localization, that is, the heterologous polypeptide contains a subcellular localization sequence (for example, a nuclear localization signal (NLS) for targeting the nucleus to maintain the sequence of the fusion protein
  • a subcellular localization sequence for example, a nuclear localization signal (NLS) for targeting the nucleus to maintain the sequence of the fusion protein
  • the nuclear export sequence (NES) outside the nucleus maintains the sequence of the fusion protein retained in the cytoplasm, and is used to target the mitochondrial mitochondrial localization signal, the chloroplast localization signal used to target the chloroplast, the Golgi localization signal, etc.
  • type V CRISPR/Cas effector proteins do not include NLS, so the protein does not target the nucleus.
  • NLS include NLS sequences derived from the following: NLS of the SV40 virus large T antigen, with the amino acid sequence PKKKRKV; NLS from the nucleoplasmic protein (for example, the nucleoplasmic protein dyad NLS having the sequence KRPAATKKAGQAKKKK); c -myc NLS has the amino acid sequence PAAKRVKLD or RQRRNELKRSP; hRNPA1 M9 NLS has the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY.
  • the heterologous polypeptide may provide a label (ie, the heterologous polypeptide is a detectable label) to facilitate tracking and/or purification (e.g., fluorescent protein: green fluorescent protein (GFP), yellow fluorescent protein (YFP), Red fluorescent protein (RFP), blue fluorescent protein (CFP), mCherry, tdTomato, etc.; histidine tag: 6 ⁇ His tag; hemagglutinin (HA) tag; FLAG tag; Myc tag; biotin tag; Avidin tags, etc.).
  • the fusion protein in some cases, may contain one or more positioning sequences or tags, and each tag or positioning sequence may be one or more repeats.
  • the parameter "identity” describes the correlation between two amino acid sequences or nucleotide sequences.
  • the degree of identity between two amino acid sequences or nucleotide sequences uses, for example, the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice etc., 2000, Trends in Genetics 16: 276-277) ), preferably the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in version 3.0.0 or higher.
  • the optional parameters used are gap penalty 10, gap extension penalty 0.5 and EBLOSUM62 replacement matrix (EMBOSS version of BLOSUM62).
  • the crRNA of the present invention refers to CRISPR RNA, which is a single-stranded guide RNA that guides the Cas protein to specifically bind to African swine fever virus genes, such as K205R, CP530R, CP204L and P72 gene sequences.
  • the crRNA of the present invention is a nucleic acid molecule that can bind to type V CRISPR/Cas effector protein (Cas12 protein, such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, etc.) to form a ribonucleoprotein complex (RNP) and target the complex To the K205 gene fragment (also called target sequence DNA).
  • Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, etc.
  • crRNA may be hybrid DNA/RNA, so that in addition to RNA bases, the crRNA also includes DNA bases.
  • the crRNA of the present invention includes a guide sequence (also referred to as a "spacer", which can hybridize to the target sequence DNA), and a constant region (a region adjacent to the guide sequence and binding to the V-type CRISPR/Cas effector protein).
  • the "constant region” may also be referred to as a "protein binding segment” herein.
  • the constant region is at the 5'end of the crRNA.
  • crRNA is a nucleic acid that can guide Cas12a to bind to target DNA.
  • target DNA and target sequence DNA have the same meaning, and both refer to the K205 gene fragment that needs to be combined and detected by the CRISPR/Cas system of the present invention.
  • Cas12a protein After crRNA binds to Cas12 protein to form a ribonucleoprotein complex, when it performs its function, Cas12a protein recognizes the protospacer-adjacent motif (PAM region) (TTTN or TTN), and performs single-base on the target sequence DNA. When the appropriate PAM region is found, the Cas12a protein will form a semi-closed R-loop (R-loop). When the correct sequence complementary to crRNA is recognized, it will be completely combined to form a closed R -Loop, and then specifically cut the target (Strohkend et al., 2018, Molecular Cell 71, 1-9).
  • PAM region protospacer-adjacent motif
  • the guide sequence in the crRNA of the present invention is complementary to the target DNA fragment (target sequence) on the African swine fever virus gene fragment, and the target sequence complementary to the crRNA needs to have a PAM region before the 5'end.
  • the length of the guide sequence is 15-28 nucleotides (nt), for example, preferably 15-26nt, 15-24nt, 15-22nt, 15-20nt, 15-18nt, 16-28nt, 16-26nt, 16-24nt, 16-22nt, 16-20nt, 16-18nt, 17-26nt, 17-24nt, 17-22nt, 17-20nt, 17-18nt, 18-26nt, 18-24nt or 18- 22nt length).
  • the length of the guide sequence is 18-24 nucleotides (nt). In some cases, the guide sequence is at least 15 nt long (e.g., at least 16, 18, 20, or 22 nt long). In some cases, the guide sequence is at least 17 nucleotides. In some cases, the guide sequence is at least 18 nucleotides. In some cases, the guide sequence is at least 20 nucleotides.
  • the guide sequence and the target sequence of the target DNA have 80% or more (eg, 85% or more, 90% or more, 95% or more, or 100%) complementarity. In some cases, the guide sequence is 100% complementary to the target sequence of the target DNA. In some cases, the target DNA contains at least 15 nucleotides (nt) that are complementary to the guide sequence of the guide RNA.
  • Cas12 proteins such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e
  • Table 1 Examples of constant regions of guide RNAs that can be used with V-type CRISPR/Cas effector proteins (Cas12 proteins, such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e) are shown in Table 1.
  • the constant region of the crRNA includes a nucleotide sequence having 70% or more identity with the constant region of any crRNA sequence shown in Table 1 (for example, 80% or more, 85 % Or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% identity).
  • the constant region of crRNA includes or has the nucleotide sequence shown in Table 1.
  • the constant region contains complementary RNA sequences that self-fold to form RNA duplexes (dsRNA).
  • dsRNA RNA duplexes
  • the constant region of crRNA is 15 or more nucleotides (nt) in length (for example, 18 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 Or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 Or more, or 35 or more nt length).
  • the constant region of crRNA is 18 or more in length.
  • the length of the constant region of crRNA ranges from 12 to 100 nt (e.g., 12 to 90, 12 to 80, 12 to 70, 12 to 60, 12 to 50, 12 to 40, 15 to 100, 15 to 90 , 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 25 To 100, 25 to 90, 25 to 80, 25 to 70, 25 to 60, 25 to 50, 25 to 40, 28 to 100, 28 to 90, 28 to 80, 28 to 70, 28 to 60, 28 to 50 , 28 to 40, 29 to 100, 29 to 90, 29 to 80, 29 to 70, 29 to 60, 29 to 50, or 29 to 40 nt). In some cases, the length of the constant region of crRNA ranges from 28 to 100 nt. In some cases, the length of the constant region at the 5'end of crRNA ranges from 28 to 40 nt.
  • the constant region of the crRNA is truncated relative to the corresponding region of the corresponding wild-type guide RNA. In some cases, the constant region of the crRNA is extended relative to the corresponding region of the corresponding wild-type crRNA. In some cases, the crRNA is 29 or more nucleotides (nt) in length (for example, 34 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more More, 65 or more, 70 or more, or length of 80 or more). In some cases, the length of crRNA is 35 or more.
  • African swine fever virus is a single-molecule linear double-stranded DNA virus that belongs to the African swine fever virus family and is currently the only member of this family.
  • There are 24 genotypes of the ASFV virus that have been discovered (Galindo & Alonso, 2017; Quembo et al., 2018), and the genome sequence has been determined by genome sequencing.
  • the method of the present invention can detect all discovered strains involving 24 ASFV gene subtypes.
  • ASFV viruses include BA71V, Ken05TK1, strain E75, Georgia 2007/1, Ken06, strain L60, Benin 971, 26544OG10, NHV, OURT 883, 47Ss2008, R35, N10, R25, R7, R8 , ASFV POL 2015 Podlaskie, Warthog, Warmbaths, Tengani 62, Pretorisuskop 96 4, Mkuzi 1979, Malawi Lil-20 1 (1983), and isolate Kenya 1950.
  • the K205R gene (GeneID: 22220430) is one of the early transcription genes in the ASFV genome, with a full length of 618 bp.
  • the pK205R protein encoded by this gene can stimulate the body to produce antibodies earlier; the present invention takes the relatively conservative fragments of the K205R gene as Target sequence to design crRNA.
  • the selected target sequence in the K205R gene is as follows:
  • Cas12a/T2-crRNA1 target sequence TCCATGGTATCGTAACGTTC (SEQ ID No: 10)
  • the target sequence in the K205R gene has a length of at least 15 bp.
  • the K205R gene fragment refers to the length of at least 15 bp in the K205R gene, preferably a sequence having a length of at least 20 bp.
  • Artificially synthesized the selected African swine fever virus K205R gene fragment sequence ie, the target sequence, 196 bp
  • cloned it into the pUC-57 plasmid (Table 2), and used the plasmid extraction kit to extract the target gene plasmid DNA as a template,- Store at 80°C for later use.
  • the probe is a small piece of single-stranded DNA fragment. Unlike ordinary probes, the sequence of the DNA fragment can be any sequence and does not need to be complementary to the target DNA.
  • the length of the single-stranded DNA probe can be 3-180 nt, and the preferred length is 5-30 nt.
  • the 5'end and 3'end of the single-stranded DNA probe are respectively labeled with a fluorescent group and a quenching group; if selected, the single-stranded DNA may be.
  • the fluorescent group can be, but not limited to, FAM (Carboxy fluorescein, green fluorescence), FITC (Fluorescein isothiocyanate), TET (Tetrachloro fluorescein, Tetrachloro fluorescein).
  • HEX hexachloro-6-methyl fluorescein, Hexachloro fluorescein
  • JOE 2,7-dimethyl-4,5-dichloro-6-carboxy fluorescein
  • rhodamine Rhodamine dyes such as R110 , TAMRA, Texas Red, etc.
  • ROX rhodamine
  • AlexaFluor dyes such as Alexa 350, Alexa 405,Alexa 430,Alexa 488,Alexa 500, Alexa 514,Alexa 532,Alexa 546,Alexa 555,Alexa 568,Alexa 594,Alexa 610,Alexa 633,Alexa 635,Alexa 647,Alexa 660,Alexa 680, Alexa 700, Alexa 750, Alexa 790
  • ATTO dyes such as ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, AT
  • the quenching group can be, but not limited to, DABCYL, TAMRA, MGB, BHQ-0, BHQ-1, BHQ-2, BHQ-3 and the like.
  • the single-stranded DNA probe is 5-FAM/TTATTAATTATA/BHQ1-3.
  • the swine fever virus DNA is extracted; this extraction process can use conventional methods in the field or conventional extraction kits, For example, TaKaRaMiniBEST virus RNA/DNA extraction kit to extract swine fever virus DNA.
  • the extracted sample DNA can be directly used for the detection of the present invention, or in the case of a small amount of sample DNA, it is amplified before the detection of the present invention is performed.
  • the nucleic acid amplification system in the present invention can be divided into two types: the traditional PCR method and the isothermal amplification RPA method.
  • PCR polymerase chain reaction
  • a recombinase polymerase amplification (RPA) reaction can be used to amplify sample DNA.
  • the RPA reaction uses a recombinase that can pair sequence-specific primers with homologous sequences in a double-stranded DNA sample. If there is a target DNA paired with the primer, the DNA amplification is started, and no other sample operations, such as thermal cycling, are required.
  • the entire RPA amplification system is a stable and dry formula, which can be safely transported without refrigeration.
  • the RPA reaction can be carried out at an isothermal temperature, and the optimal reaction temperature is 37-42°C. In order to amplify the sequence containing the DNA of the sample to be tested, sequence-specific primers can be designed.
  • RNA polymerase promoter such as a T7 promoter
  • an RNA polymerase promoter is added to one of the primers. This results in an amplified double-stranded DNA product that contains both the target DNA sequence and the RNA polymerase promoter.
  • the detection system includes crRNA, Cas12a protein, single-stranded DNA nucleic acid probe, buffer, etc., and incubate at 20-42°C for 1 minute to 24 Hours, the temperature is preferably 37° C., the incubation time is preferably 5 minutes to 3 hours, more preferably 15 minutes to 1 hour, and then the solution is subjected to fluorescence detection.
  • the sample DNA can be specifically amplified first.
  • the detection system includes crRNA, Cas12a protein, single-stranded DNA nucleic acid probe, buffer, etc., and then performs fluorescence detection on it.
  • PCR amplification system containing the DNA of the sample to be tested, including sample DNA, PCR primers, PCR enzyme mixture, PCR buffer, etc.; after that, the PCR amplification product is added to the detection system, which includes crRNA, Cas12a protein, Single-stranded DNA nucleic acid probes, buffers, etc., and then fluorescence detection.
  • the invention finally judges whether the African swine fever virus exists in the sample by detecting the change of the fluorescence intensity of the solution system.
  • the fluorescence detection system of the present invention needs the following modules: 1) Temperature control module, the temperature control range is 0-100°C, the better case temperature control range is 25-50°C, and the better case temperature control is constant at 37°C 2) Fluorescence detection module, excitation light wavelength is 490nm, emission light wavelength is 520nm; or excitation light wavelength is 535nm, emission light wavelength is 560nm; 3) Timing detection function, can set fluorescence every 0.5-120 minutes For testing, it is better to perform a test every 2-15 minutes, in a better case it is to perform a test every 2-5 minutes, the duration is 10 minutes to 3 hours, and the better case is 15 minutes to 2 hours.
  • the fluorescence detection system may be BioTekCytation 3; in certain exemplary embodiments, the fluorescence detection system may be ThermoVarioskan TM LUX; in certain exemplary embodiments, the fluorescence detection system may be fluorescence For quantitative PCR, in certain exemplary embodiments, the fluorescence detection system may be an Applied Biosystems TM 7500 Real-Time PCR System.
  • Example 1 RPA reaction using synthetic K205R gene plasmid DNA as template
  • K205R gene segment SEQ ID NO.11 was directly synthesized and cloned into pUC-57 vector ( GenScriptCat. No. SD1176), synthetic plasmid pUC-57-K205R.
  • RPA primers According to the design requirements of RPA primers, RPA primers contain up to two variable base positions, the designed upstream primer RPA-F (SEQ ID NO.35) and downstream primer RPA-R( SEQ ID NO. 36) See Table 3 for the sequence;
  • RPA reaction and agarose gel electrophoresis Dilute the above designed and synthesized primers to a final concentration of 10uM, and add the upstream primer RPA-F (SEQ ID NO.35) and downstream to the reaction system containing the lyophilized powder of RPA reaction enzyme primer RPA-R (SEQ ID NO.36) each 2.4ul, RPA buffer (RPA provided in the kit) 29.5ul, 10 4 (represented by 1E4) and 108 (indicated by 1E8) a different copy number of pUC-57- K205R plasmid DNA, 3.5 ⁇ l 280 mMMgAc ion, add ddH 2 O to make up to 50ul, and react at 37°C for 15 min.
  • Example 2 after T1-crRNA1 forms a complex with the Cas12a protein, the K205R gene is specifically cut and the ssDNA fluorescent probe is subjected to fluorescence detection of non-specific cut.
  • T1-crRNA1 was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • the RNA sequence (SEQ ID NO.37) of T1-crRNA1 is shown in Table 3.
  • the synthesized RNA was diluted to 20uM and stored at -20°C for later use.
  • Cas12a-crRNA binding reaction In 10ul buffer (40mM Tris-HCl; 60mM NaCl; 6 mM MgCl 2 ) add 10ul LbCas12a protein (SEQ ID No: 1, purchased from Integrated DNA Technologies) at a concentration of 40uM and a concentration of 20uM 20ul of crRNA (T1-crRNA1), prepared into a system with a final concentration of Cas12a-crRNA of 10uM, and incubate at room temperature for 20min;
  • 10ul buffer 40mM Tris-HCl; 60mM NaCl; 6 mM MgCl 2
  • 10ul LbCas12a protein SEQ ID No: 1, purchased from Integrated DNA Technologies
  • RNA sequence of T2-crRNA1 (SEQ ID NO.38) instead of T1-crRNA1, and operate under the same experimental conditions as above.
  • the results in Fig. 2 show that for a DNA concentration of 100 nM, the fluorescence signals of the T1-crRNA1 and T2-crRNA1 groups can show a significant increase in about 15 minutes, indicating that efficient cutting is achieved.
  • Cas12a-crRNA binding reaction add 10ul LbCas12a protein (purchased from Integrated DNA Technologies) (40uM) and 20ul T1-crRNA1 (20uM) in 10ul buffer (40mM Tris-HCl; 60mM NaCl; 6mM MgCl 2 ), Configured into a system where the final concentration of Cas12a-crRNA is 10uM, incubate at room temperature for 20min;
  • Step 2) Preparation of the detection system: Add 5ul of Cas12a-crRNA complex in step 1) to the detection system, and add 1ul of pUC-57-K205R plasmid DNA (10 7 , 10 8 , 10 9 , 10 10 copies, respectively denoted by E7, E8, E9, E10), 44ul of buffer (40mM Tris-HCl; 60mM NaCl; 6mM MgCl2), mixed and added to the DNaseAlert TM Substrate ( Purchased from Integrated DNA Technologies), pipetting and mixing;
  • Example 4 A method for detecting African swine fever virus based on the combination of Cas12a and RPA, using a synthetic plasmid DNA with a partial conserved region sequence of the African swine fever virus K205R gene as a target for detection
  • RPA pre-amplification reaction Add 2.4ul each of the upstream primer RPA-F (SEQ ID NO.35) and downstream primer RPA-R (SEQ ID NO.36) into the reaction system containing the lyophilized powder of RPA reaction enzyme.
  • RPA buffer (provided in the RPA kit, purchased from TwistDx) 29.5ul, add 1ul 10-fold dilutions of pUC-57-K205R plasmid DNA (10, 10 2 , 10 4 , 10 8 copies per microliter) : In the figure represented by E1, E2, E4, E8 respectively), 3.5 ⁇ l 280 mMMgAc ion, add ddH 2 O to make up to 50ul, and react at 37°C for 15 min.
  • Cas12a-crRNA binding reaction Add 10ul LbCas12a protein (Integrated DNA Technologies) (40uM) and 20ul T1-crRNA1 (20uM) in 10ul buffer (40mM Tris-HCl; 60mM NaCl; 6mM MgCl2) to configure Cas12a -crRNA final concentration is 10 ⁇ M system, incubate at room temperature for 20min.
  • 10ul buffer 40mM Tris-HCl; 60mM NaCl; 6mM MgCl2

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Abstract

本发明公开了一种快速检测非洲猪瘟病毒的方法，尤其是一种基于基因编辑***的核酸检测方法，具体是基于CRISPR-Cas12a核酸蛋白复合物的病毒核酸快速检测方法。本发明基于Cas12a在crRNA的指导下进行特异性切割ASFV基因DNA的同时，能产生非特异性的ssDNA切割活性的特性进行核酸检测。本发明适合于非洲猪瘟病毒的快速诊断和早期筛查。

Description

一种检测非洲猪瘟病毒的方法和试剂盒 技术领域
本发明属于病毒的检测领域，尤其涉及检测非洲猪瘟病毒的体系，检测方法及其试剂盒。本发明能用在非洲猪瘟病毒或者其它可感染猪的病毒检测中的应用。
背景技术
非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus，ASFV)引起的一种急性的具有出血热等临床症状的高度传染性疾病，致死率高于90％。ASFV是一种线状双链DNA病毒，基因组长约170kb～193kb，可以编码150～200种蛋白。ASFV主要在野猪、家猪和软蜱之间循环感染传播，由于其极高的传染性和致死性，对受灾地区的养猪业造成严重的经济负担。ASF被世界卫生组织(OIE)列为法定报告动物疫病之一，在中国被列为一类动物传染病。ASF最早于上世纪60年代在非洲发现，之后传播蔓延到欧洲、美洲等大陆，现已发现60多种不同的ASFV病毒株，根据P72基因目前可以分为24种基因型。2018年8月，我国沈阳市确诊发生首例ASF疫情，约400例感染猪在疫情发生后的一个月内全部死亡。随后，我国多省和地区相继发现ASF疫情，对于ASF疫情的控制已刻不容缓。
由于目前尚未发现有效的ASF疫苗和治疗方法，快速准确的诊断和筛查方法用于疫情的早期甚至是潜伏期的发现对于疫疾的防控显得极为关键。对于ASFV的检验检疫和快速诊断，市场上并没有一个标准方法和成熟的产品，主要是基于临床表型和实验室方法进行诊断和确诊。现有的实验室诊断方法主要集中在病毒分离鉴定、抗原抗体免疫检测和病毒核酸PCR检测等三大方向。病毒分离鉴定的方法属于传统的金标准诊断方法，但是流程繁琐、耗时耗力，通常被实验室用做验证性诊断方法。基于抗原抗体的免疫检测包括直接或间接荧光抗体检测和酶联免疫吸附实验检测，虽然流程简单，但是灵敏度不够，容易出现假阴性和漏检。PCR法具备一定的灵敏度和特异性，是市面上较为流行的检测方法，包括OIE推荐的用于实验室常规诊断的常规PCR和荧光实时定量PCR法(Agüero M,et al.J ClinMicrobiol 2003,41:4431–4434；)。但是，有研究报道报道显示，OIE推荐的方法中引 物和病毒靶基因之间的核苷酸错配会导致PCR的敏感性和特异性降低(Gallardo C et al.J ClinMicrobiol.2015 Aug；53(8):2555-65)。随着PCR技术的不断更新和对ASFV更多的研究报道涌现，一些新的ASFV检测技术和方法也逐渐显现。Wang等人的研究报道了基于重组酶聚合酶扩增(recombinase polymerase amplification，RPA)技术的ASFV快速检测方法，可以在10min内检测出100拷贝左右的含有ASFV P72基因区段的重组质粒DNA分子(Can J Vet Res.2017 Oct；81(4):308-312)。2018年，Massimo Biagetti等人也报道了基于生物传感器进行ASFV快速检测的方法(Talanta.2018 Jul 1；184:35-41)。虽然这些技术报道结果的灵敏度较高，但是这些技术距离实际应用还有一段路要走，并且不够便捷和高效，因此市场上急需一些新的便捷、高效的检测方法用于ASFV的快速检测和早期筛查。
2012年，科学家们首次发现了成簇规律间隔短回文重复序列(CRISPR:Clustered Regularly Interspaced Short Palindromic Repeats)基因编辑技术。CRISPR-Cas***是微生物体内的一种RNA指导的适应性免疫***，当病毒侵入细菌时，细菌能够捕捉到外来遗传物质的片段并且将它们整合到自身基因组中的CRISPR序列中。CRISPR序列转录生成的CRISPR RNA(crRNA)能够与CRISPR结合蛋白(Cas核酸酶)相结合，通过与靶点核酸序列进行碱基配对为Cas核酸酶提供结合与切割特异性(Cong et al.Science 2013.339:819-823)。Cas9核酸酶是第一个被广泛认可并应用于基因组编辑的Cas蛋白，但是目前科学家们已经将Cas蛋白的范围扩展到更多其它类型的Cas蛋白中，比如新近报道的Cas13a，Cas12a以及Cas14a蛋白等。Cas13a核酸酶在crRNA作用下切割特异性靶标RNA，同时激活自身核酸酶的活性对非特异性的RNA进行切割。
张锋团队等基于Cas13a和RPA等温扩增技术，开发了SHERLOCK***用于核酸检测，对Zika和Dengue病毒进行快速精准检测(Zhang et al.Science.2017 Apr 28；356(6336):438–442)。詹尼弗等研究组发现Cas12a以及其他V型CRISPR-Cas12酶具有目标激活的非特异性ssDNase切割的特性，通过将Cas12a与等温扩增相结合创建了DETECTR核酸检测方法，该方法实现了DNA检测的超高的灵敏度，能够快速、专一地检测患者样本中的人***瘤病毒(Doudna JA et al.Science.2018 Apr 27；360(6387):436-439)。
目前非洲猪瘟病毒的防控遭遇重大挑战，在中国乃至全球，并没有一款产品能够便捷、高效的用于ASFV的快速检测和早期筛查，尤其是没有基于基因编辑酶技术开发的诊断和早期筛查型产品。
发明内容
本发明的目的在于提供一种基于Cas12蛋白的非洲猪瘟病毒的核酸快速检测体系和检测方法。
本发明提供了如下技术方案：
1.一种用于检测非洲猪瘟病毒的核酸检测体系，包含：
a)Cas12蛋白；b)一个或多个crRNA，其中crRNA能与对应的非洲猪瘟病毒的基因片段结合；和c)单链DNA荧光探针。
2.技术方案1所述的检测体系，其中所述的非洲猪瘟病毒的基因为K205R(全长序列为GeneID：22220430)、CP530R(全长序列为GeneID：22220323)、CP204L(全长序列为GeneID：22220322)和P72(全长序列为GeneID：22220311)，优选为K205R基因。
3.技术方案1或2所述的检测体系，其中所述的Cas12蛋白选自Cas12a，Cas12b或Cas12c。
4.技术方案3所述的检测体系，其中所述的Cas12a蛋白选自FnCas12a、AsCas12a、LbCas12a、Lb5Cas12a、HkCas12a、OsCas12a、TsCas12a、BbCas12a、BoCas12a或Lb4Cas12a。
5.技术方案4所述的检测体系，其中所述的Cas12a蛋白具有SEQIDNo：1所示的序列；或与SEQIDNo：1所示的序列具有至少80％同一性的序列；或与SEQIDNo：1所示的序列具有一个或几个氨基酸的取代、缺失和***的Cas12a蛋白的变体。
6.技术方案5所述的检测体系，其中所述的Cas12a蛋白与SEQIDNo：1所示的序列具有至少83％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％同一性的序列。
7.前述任一项技术方案所述的检测体系，其中所述的非洲猪瘟病毒的基因片段，例如K205R基因片段具有至少15bp的长度，优选具有至少20bp的长度。
8.前述任一项技术方案所述的检测体系，其中所述的crRNA的指导序列具有15nt-28nt的长度。
9.前述任一项技术方案所述的检测体系，其中所述的crRNA具有29-100nt的长度。
10.技术方案9所述的检测体系，其中所述的crRNA具有40-50nt的长度。
11.前述任一项技术方案所述的检测体系，其中多个单链crRNA结合同一个非洲猪瘟病毒的基因片段，例如K205R基因片段。
12.前述任一项技术方案所述的检测体系，其中多个单链crRNA结合不同的非洲猪瘟病毒的基因片段，例如K205R基因片段。
13.前述任一项技术方案所述的检测体系，其中所述的单链DNA荧光探针的5’端和3’端分别标记荧光基团和猝灭基团。
14.前述任一项技术方案所述的检测体系，还进一步包含非洲猪瘟病毒的核酸的扩增体系。
15.技术方案14所述的检测体系，其中所述的扩增体系为重组酶聚合酶扩增(RPA)体系。
16.技术方案14所述的检测体系，其中所述的扩增体系为PCR扩增体系。
17.前述任一项技术方案所述的检测体系，其中所述的非洲猪瘟病毒的核酸来自于猪的组织，该组织优选是全血、猪血浆、猪血清、猪尿液、猪唾液或猪口腔粘膜。
18.一种检测非洲猪瘟病毒的核酸检测方法，包含：
a.将核酸样品与技术方案1-17任一项所述的检测体系在20℃-42℃孵育，检测荧光强度的变化；
b.所述的核酸样品是从非洲猪瘟病毒中提取的样品DNA，或者是从非洲猪瘟病毒中提取的样品DNA经过扩增而得到的靶标DNA。
19.技术方案17或18述的检测方法，其中，所述的DNA扩增为重组酶聚合酶扩增(RPA)或者PCR扩增。
20.技术方案18或19所述的检测体系，其中所述的核酸样品来自于猪的组织，该组织优选是全血、猪血浆、猪血清、猪尿液、猪唾液或猪口腔粘膜。
本发明提供的基于基因编辑酶的快速检测非洲猪瘟病毒的方法，包括基本检测体系和扩增后检测体系，与现有技术相比，具有以下一项或多项优势：
-高灵敏度：应用本发明可以实现对aM级别的DNA检测，检测限低至10个拷贝，除了ASFV快速定性检测外，也可以实现ASFV的早期筛查，因假阴性造成的漏检率极低；
-高特异性：酶标仪检测时间短至15min的情况下，分析荧光数值变化，同时结合多重crRNA靶点的对比结果，最大可能降低假阳性，增强特异性；
-快速：本发明可以短至半小时内完成检测；
-便捷：实现单管多个试剂的一步法检测，操作方便，步骤简单；
-通用性：应用本发明可以实现ASFV等DNA病毒的快速检测。
附图说明
图1：本发明实施例提供的以合成K205R基因质粒DNA为模板进行RPA反应的产物在2％的琼脂糖凝胶电泳结果示意图(有T7和无T7)；
图2：本发明实施例提供的T1-crRNA1和T2-crRNA1与Cas12a蛋白形成复合体后对K205R基因进行特异性切割和ssDNA荧光探针进行非特异性切割的荧光检测结果示意图；
图3：本发明实施例提供对不同初始量的K205R基因进行Cas12a基本检测的荧光检测结果示意图；
图4：本发明实施例提供的基于Cas12a和RPA的非洲猪瘟病毒检测方法的灵敏度示意图(连续不同时间和某个时间)；
图5：另一个基因靶标的Cas12a和RPA联合检测灵敏度示意图。
本发明的详述
本发明在此通过对使用下述定义和实施例的引用进行详细描述。所有在本文中提及的专利和公开文献的内容，包括在这些专利和公开中披露的所有序列，明确地通过提述并入本文。
CRISPR/Cas体系
CRISPR是指成簇的、规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats)，该序列是许多原核生物的免疫***。
Cas蛋白是指CRISPR/Cas效应蛋白，目前研究发现，CRISPR-Cas***主要有两大类，第一类是包含多亚基蛋白效应子复合物的Class1和单亚基蛋白效应子复合物的Class2，其中Class1CRISPR-Cas***在细菌和古生菌(包括所有超嗜热菌)中最为常见，该类蛋白约占所有已鉴定的CRISPR-Cas蛋白的90％(Makarova KS,et al.An updated evolutionary classification of CRISPR–Cas systems.Nat.Rev.Microbiol.2015；13:722–736)。Class 2 CRISPR-Cas***几乎只存在于细菌中，该***可用的Cas蛋白主要包括II，V，VI型效应子蛋白，约占CRISPR-Cas蛋白的10％，主要有常用的Cas9蛋白(II型)，以及新近被发现的Cas12(V型)、Cas13(VI)型和Cas14(V型)蛋白等(Chylinski K et al.Nucleic Acids Res. 2014；42:6091–6105；Shmakov S,et al.Mol.Cell.2015；60:385–397；Sergey Shmakov et al.Nat Rev Microbiol.2017 March；15(3):169–182；DoudnaJAet al.Science.2018 Nov 16；362(6416):839-842)。由于Class 2 CRISPR-Cas***相对简单的效应复合物构成，使其成为最热的用于科学研究的基因编辑工具之一。
本申请中“CRISPR/Cas体系”和“CRISPR体系”是相同的意思。
Cas蛋白
本发明技术方案所涉及的Cas蛋白为Cas12(V型CRISPR/Cas效应蛋白)蛋白家族。Cas12家族包括Cas12a、Cas12b(C2c1)、Cas12c(C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12g、Cas12h和Cas12i等；Cas12的定义参见文献Koonin et al.,CurrOpinMicrobiol.2017 June；37:67-78:“Diversity,classification and evolution of CRISPR-Cas systems”，该文献的内容作为本专利申请的一部分。这些Cas蛋白可以来源于不同的菌属，其酶活性也可能存在不同。
在具体的实施方式中，本发明的Cas12蛋白是Cas12a蛋白。Cas12a蛋白(旧称“Cpf1”)是指crRNA依赖的内切酶，它是CRISPR***分类中V型(type V)的酶。本发明的Cas12a蛋白可以是不同种属来源的Cas12a蛋白，例如FnCas12a、AsCas12a、LbCas12a、Lb5Cas12a、HkCas12a、OsCas12a、TsCas12a、BbCas12a、BoCas12a和Lb4Cas12a中的一种。所述Cas12a蛋白优选为LbCas12a；更优选是图5所示序列的Cas12a(SEQIDNo：1)，或与SEQIDNo：1具有至少80％，83％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％同一性的Cas12a，或者是与SEQIDNo：1具有一个或几个氨基酸的缺失、取代或添加的Cas12a的变体，并且该变体仍然具有Cas12a的功能。在具体实施方式中，一个或几个氨基酸的缺失、取代或添加的Cas12a的变体是指1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸的缺失、取代或添加。
在一些情况下，Cas12蛋白，例如Cas12a，Cas12b，Cas12c，Cas12d，Cas12e为融合蛋白。在一些情况下，异源多肽(融合配体)提供亚细胞定位，即异源多肽含有亚细胞定位序列(例如，用于靶向细胞核的核定位信号(NLS)，用于保持融合蛋白的序列在细胞核外的核输出序列(NES)，保持融合蛋白保留在细胞质中的序列，用于靶向线粒体的线粒体定位信号，用于靶向叶绿体的叶绿体定位信号，高尔基体定位信号等)。在一些情况 下，V型CRISPR/Cas效应蛋白(例如，Cas12蛋白)不包括NLS，因此蛋白质不靶向细胞核。NLS的非限制性实例包括衍生自以下的NLS序列：SV40病毒大T抗原的NLS，具有氨基酸序列PKKKRKV；来自核质蛋白的NLS(例如，具有序列KRPAATKKAGQAKKKK的核质蛋白二分体NLS)；c-myc NLS具有氨基酸序列PAAKRVKLD或RQRRNELKRSP；hRNPA1 M9 NLS具有序列NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY。在一些情况下，异源多肽可以提供标签(即，异源多肽是可检测的标记)以便于追踪和/或纯化(例如，荧光蛋白：绿色荧光蛋白(GFP)，黄色荧光蛋白(YFP)，红色荧光蛋白(RFP)，蓝色荧光蛋白(CFP)，mCherry，tdTomato等；组氨酸标签：6×His标签；血凝素(HA)标签；FLAG标签；Myc标签；生物素标签；连霉亲和素标签等)。所述融合蛋白，在一些情况下，包含的定位序列或标签可以是一种或多种，每种标签或定位序列，可以是一个或者多个重复。
同一性
由参数“同一性”描述两个氨基酸序列或核苷酸序列之间的相关性。
就本发明而言，两个氨基酸序列或核苷酸序列之间的同一性程度使用如EMBOSS软件包(EMBOSS：The European Molecular Biology Open Software Suite，Rice等，2000，Trends in Genetics 16：276-277)的Needle程序，优选3.0.0版或更高版本中执行的Needleman-Wunsch算法(Needleman和Wunsch，1970，J.Mol.Biol.48：443-453)来确定。使用的任选参数为缺口罚分(gap penalty)10，缺口延伸罚分(gap extension penalty)0.5和EBLOSUM62取代矩阵(BLOSUM62的EMBOSS版)。使用Needle标记为“最长同一性(longest identity)”(使用-nobrief选项获得)的输出结果作为百分比同一性，并计算如下：
(相同的残基×100)/(比对的长度-在比对中的空位总数)。
crRNA
本发明的crRNA是指CRISPR RNA，是引导Cas蛋白特异性结合非洲猪瘟病毒的基因，例如K205R、CP530R、CP204L和P72基因序列的单链指导RNA。本发明的crRNA是一种核酸分子，能结合V型CRISPR/Cas效应蛋白(Cas12蛋白，例如Cas12a，Cas12b，Cas12c，Cas12d，Cas12e等)形成核糖核蛋白复合物(RNP)，并将复合物靶向K205基 因片段(也称为靶序列DNA)。在一些实施方式中，crRNA可以是杂合DNA/RNA，使得crRNA中除RNA碱基外也包括DNA碱基。本发明的crRNA包括指导序列(也称为“spacer”，可与靶序列DNA杂交)，和恒定区(与指导序列相邻并与V型CRISPR/Cas效应蛋白结合的区域)。“恒定区”在本文中也可称为“蛋白质结合区段”。在一些实施例中，例如对于Cas12a，恒定区是在crRNA的5'端。在本发明中，crRNA是可以引导Cas12a结合到靶标DNA的核酸。本申请中“靶标DNA”和“靶序列DNA”是相同的意思，都是指本发明的CRISPR/Cas体系需要结合和检测的K205基因片段。
crRNA与Cas12蛋白结合成核糖核蛋白复合体后，在行使功能时，Cas12a蛋白识别原型间隔子邻近基序(protospacer-adjacent motif,PAM区)(TTTN或TTN)，通过对靶序列DNA进行单碱基的识别，当寻找到合适的PAM区后，Cas12a蛋白会形成一种半封闭的R-环(R-loop)，当识别到与crRNA互补的正确序列后，才会完全结合形成封闭的R-环，然后对靶标进行特异性的切割(Strohkendl et al.,2018,Molecular Cell 71,1–9)。
本发明crRNA中的指导序列与非洲猪瘟病毒基因片段上的靶标DNA片段(靶序列)互补，与crRNA互补的靶序列需要在5’端之前具有PAM区。在具体的实施方案中，指导序列的长度为15-28个核苷酸(nt)，例如优选为15-26nt,15-24nt,15-22nt,15-20nt,15-18nt,16-28nt,16-26nt,16-24nt,16-22nt,16-20nt,16-18nt,17-26nt,17-24nt,17-22nt,17-20nt,17-18nt,18-26nt,18-24nt或18-22nt长度)。在具体实施例中，指导序列的长度为18-24个核苷酸(nt)。在一些情况下，指导序列至少15nt长(例如，至少16,18,20或22nt长)。在某些情况下，指导序列至少为17个核苷酸。在某些情况下，指导序列至少为18个核苷酸。在某些情况下，指导序列至少为20个核苷酸。
在一些情况下，指导序列与靶DNA的靶序列具有80％或更多(例如，85％或更多，90％或更多，95％或更多，或100％)的互补性。在一些情况下，指导序列与靶DNA的靶序列100％互补。在一些情况下，靶DNA包含与指导RNA的指导序列互补的至少15个核苷酸(nt)。
可以与V型CRISPR/Cas效应蛋白(Cas12蛋白，例如Cas12a，Cas12b，Cas12c，Cas12d，Cas12e)一起使用的指导RNA的恒定区的实例如表1所示。
表1：crRNA的恒定区
Figure PCTCN2020093456-appb-000001
Figure PCTCN2020093456-appb-000002
在具体实施方式，所述crRNA包括的恒定区与表1中所示的任何一种crRNA序列的恒定区具有70％或更高同一性的核苷酸序列(例如，80％或更多，85％或更多，90％或更多，95％或更多，98％或更多，99％或更多，或100％同一性)。在某些具体实施方式中，crRNA的恒定区包括或者具有表1中所示的核苷酸序列。
在一些具体实施方式中，恒定区包含互补的RNA序列，其通过自折叠形成RNA双链体(dsRNA)。
在一些情况下，crRNA的恒定区长度为15或更多核苷酸(nt)(例如，18或更多，20或更多，21或更多，22或更多，23或更多，24或更多，25或更多，26或更多，27或更多，28或更多，29或更多，30或更多，31或更多，32或更多，33或更多，34或更多，或35或更多nt长度)。在一些情况下，crRNA的恒定区长度为18或更多。在一些情况下，crRNA的恒定区的长度范围为12至100nt(例如，12至90,12至80,12至70,12至60,12至50,12至40,15至100,15至90,15至80,15至70,15至60,15至50,15至40,20至100,20至90,20至80,20至70,20至60,20到50,20到40,25到100,25到90,25到80,25到70,25到60,25到50,25到40,28到100,28到90,28到80,28到70,28至60,28至50,28至40,29至100,29至90,29至80,29至70,29至60,29至50，或29至40nt)。在一些情况下，crRNA的恒定区的长度范围为28至100nt。在一些情况下，crRNA5'端的恒定区域的长度范围为28至40nt。
在一些情况下，crRNA的恒定区相对于相应野生型指导RNA的相应区域被截短。在一些情况下，crRNA的恒定区相对于相应野生型crRNA的相应区域延长。在一些情况下，crRNA的长度为29或更多个核苷酸(nt)(例如，34或更多，40或更多，45或更多，50 或更多，55或更多，60或更多，65或更多，70或更多，或长度为80或更多)。在某些情况下，crRNA的长度为35或更多。
非洲猪瘟病毒
非洲猪瘟病毒(ASFV)是单分子线形双链DNA病毒，属于非洲猪瘟病毒科，是目前该科的唯一成员。已发现的ASFV病毒有24种基因亚型(Galindo&Alonso,2017；Quembo et al.,2018)，且都经过基因组测序确定了基因组序列。本发明的方法能检测出所有已发现的涉及24种ASFV基因亚型的毒株。
该24种已知亚型的ASFV病毒包括，BA71V，Ken05TK1，strain E75，Georgia 2007/1，Ken06，strain L60，Benin 971，26544OG10，NHV，OURT 883，47Ss2008，R35，N10，R25，R7，R8，ASFV POL 2015 Podlaskie，Warthog，Warmbaths，Tengani 62，Pretorisuskop 96 4，Mkuzi 1979，Malawi Lil-20 1(1983)，以及isolate Kenya 1950。
K205R基因
K205R基因(GeneID：22220430)是ASFV基因组中早期转录基因中的一个，全长为618 bp长度，该基因编码的pK205R蛋白能够较早刺激机体产生抗体；本发明以K205R基因中相对保守的片段作为靶点序列来设计crRNA。
在具体的实施方式中，所选取的K205R基因中的靶点序列如下：
Cas12a/T1-crRNA1靶点序列CAAGACCTGCTTTCAGCAGT(SEQ ID No:9)
Cas12a/T2-crRNA1靶点序列TCCATGGTATCGTAACGTTC(SEQ ID No:10)
在具体的实施方式中，K205R基因中的靶点序列具有至少15bp的长度。K205R基因片段是指K205R基因中的至少15bp的长度，优选具有至少20bp的长度的序列。人工合成所选取得非洲猪瘟病毒K205R基因片段序列(即，靶点序列，196 bp)并克隆至pUC-57质粒中(表2)，利用质粒提取试剂盒提取目的基因质粒DNA为模板，-80℃保存备用。
表2
Figure PCTCN2020093456-appb-000003
Figure PCTCN2020093456-appb-000004
单链DNA荧光探针
探针是一小段单链DNA片段，与通常探针不同的是，该DNA片段的序列可以是任意序列，并不需要与靶标DNA互补。所述的单链DNA探针的长度可以是3-180nt，优选的长度为5-30nt。
所述的单链DNA探针的5’端和3’端分别标记荧光基团和猝灭基团；所选的，所述的单链DNA可以是。所述的荧光基团可以为但不限于FAM(羧基荧光素，Carboxy fluorescein，绿色荧光)、FITC(异硫氰酸荧光素，Fluorescein isothiocyanate)、TET(四氯-6-羧基荧光素，Tetrachloro fluorescein)、HEX(六氯-6-甲基荧光素，Hexachloro fluorescein)、JOE(2,7-二甲基-4,5-二氯-6-羧基荧光素)、罗丹明类(Rhodamine染料如R110，TAMRA、Texas Red等)、ROX、AlexaFluor染料(如Alexa
Figure PCTCN2020093456-appb-000005
350,Alexa
Figure PCTCN2020093456-appb-000006
405,Alexa
Figure PCTCN2020093456-appb-000007
430,Alexa
Figure PCTCN2020093456-appb-000008
488,Alexa
Figure PCTCN2020093456-appb-000009
500,Alexa
Figure PCTCN2020093456-appb-000010
514,Alexa
Figure PCTCN2020093456-appb-000011
532,Alexa
Figure PCTCN2020093456-appb-000012
546,Alexa
Figure PCTCN2020093456-appb-000013
555,Alexa
Figure PCTCN2020093456-appb-000014
568,Alexa
Figure PCTCN2020093456-appb-000015
594,Alexa
Figure PCTCN2020093456-appb-000016
610,Alexa
Figure PCTCN2020093456-appb-000017
633,Alexa
Figure PCTCN2020093456-appb-000018
635,Alexa
Figure PCTCN2020093456-appb-000019
647,Alexa
Figure PCTCN2020093456-appb-000020
660,Alexa
Figure PCTCN2020093456-appb-000021
680,Alexa
Figure PCTCN2020093456-appb-000022
700,Alexa
Figure PCTCN2020093456-appb-000023
750,Alexa
Figure PCTCN2020093456-appb-000024
790)、ATTO染料(如ATTO 390,ATTO 425,ATTO 465,ATTO 488,ATTO 495,ATTO 514,ATTO 520,ATTO 532,ATTO Rho6G,ATTO 542,ATTO 550,ATTO 565,ATTO Rho3B,ATTO Rho11,ATTO Rho12,ATTO Thio12,ATTO Rho101,ATTO 590,ATTO 594,ATTO Rho13,ATTO 610,ATTO 620,ATTO Rho14,ATTO 633,ATTO 647,ATTO 647N,ATTO 655,ATTO Oxa12,ATTO 665,ATTO 680,ATTO 700,ATTO 725,ATTO 740)、DyLight染料、cyanine染料(如Cy2,Cy3,Cy3.5,Cy3b,Cy5,Cy5.5,Cy7,Cy7.5)、FluoProbes染料、SulfoCy染料、Seta染料、IRIS染料、SeTau染料、SRfluor染料、Square染料等。所述的淬灭基团可以为但不限于DABCYL、TAMRA、MGB、BHQ-0、BHQ-1、BHQ-2、BHQ-3等。例如，所述的单链DNA探针为5-FAM/TTATTAATTATA/BHQ1-3。
样品DNA获得及扩增
获取猪的组织，例如全血、猪血浆、猪血清、猪尿液、猪唾液、猪口腔粘膜之后，提取猪瘟病毒DNA；该提取过程可以采用本领域常规的方法或者常规的提取试剂盒，例如TaKaRaMiniBEST病毒RNA/DNA提取试剂盒来提取猪瘟病毒DNA。获得提取的样品DNA可以直接用于本发明的检测，或者在样品DNA量少的情况下，先经过扩增再进行本发明所述的检测。
本发明中的核酸扩增体系可分为两种：传统PCR方法和等温扩增RPA法。
在某些示例性实施方案中，传统聚合酶链式反应(PCR)可用于扩增样品DNA。将待检测样品DNA序列用设计的引物扩增后，获得靶标DNA，通过Cas12a检测体系对靶标DNA进行检测。
在某些示例性实施方案中，重组酶聚合酶扩增(RPA)反应可用于扩增样品DNA。RPA反应使用能够将序列特异性引物与双链DNA样品中的同源序列配对的重组酶。如果存在与引物配对的靶标DNA，则启动DNA扩增，不需要其他样品操作，例如热循环。整个RPA扩增***为稳定干燥配方，可以安全运输而无需冷藏。RPA反应可以在等温温度下进行，最佳反应温度为37-42℃。为了扩增包含待检测样品DNA的序列，可以设计序列特异性引物。然后可以通过Cas12a检测体系对扩增得到的靶标DNA进行检测。在某些具体实施方式中，将RNA聚合酶启动子(例如T7启动子)添加至其中一个引物。这导致扩增的双链DNA产物，同时包含靶标DNA序列和RNA聚合酶启动子。
非洲猪瘟病毒的核酸检测方法
从猪的组织中获取样品DNA之后，将待样品DNA加入检测体系中，检测体系包括crRNA、Cas12a蛋白、单链DNA核酸探针、缓冲液等，在20-42℃条件下孵育1分钟到24小时，所述温度优选为37℃，所述孵育时间优选为5分钟到3小时，更优选为15分钟到1小时，然后对溶液进行荧光检测。
在样品DNA的量不足以用于上述核酸检测时，可以先对样品DNA进行特异性扩增。可以首先建立含有待检样品DNA的RPA扩增体系，包括样品DNA、RPA引物、RPA酶混合物(其中包括重组酶和聚合酶)、MgOAc、RPA缓冲液等；之后，将RPA扩增产物加入检测体系中，检测体系包括crRNA、Cas12a蛋白、单链DNA核酸探针、缓冲液等，然后对其进行荧光检测。
也可以建立含有待检样品DNA的PCR扩增体系，包括样品DNA、PCR引物、PCR酶混合物、PCR缓冲液等；之后，将PCR扩增产物加入检测体系中，检测体系包括crRNA、Cas12a蛋白、单链DNA核酸探针、缓冲液等，然后对其进行荧光检测。
荧光检测
本发明最后通过检测溶液体系荧光强度的变化来判断样品中是否存在非洲猪瘟病毒。
本发明中的荧光检测***需要有以下几个模块：1)温度控制模块，温度控制范围为0-100℃，更优情况温度控制范围为25-50℃，更优情况温度控制在恒定37℃；2)荧光检测模块，激发光波长为490nm，发射光波长为520nm；或者激发光波长为535nm，发射光波长为560nm；3)定时检测功能，能每隔0.5-120分钟对设定的荧光进行检测，更优情况为每隔2-15分钟进行一次检测，更优情况为2-5分钟进行一次检测，持续时间为10分钟-3小时，更优情况为15分钟-2小时。
在某些示例性实施方案中，荧光检测***可以是BioTekCytation 3；在某些示例性实施方案中，荧光检测***可以是ThermoVarioskan TM LUX；在某些示例性实施方案中，荧光检测***可以是荧光定量PCR，在某些示例性实施方案中，荧光检测***可以是Applied Biosystems TM 7500 Real-Time PCR System。
实施例
实施例仅为举例说明，不旨在对本发明造成任何方式上的限制。
缩写词意义如下：“h”指小时，“min”指分钟，“s”指秒，“d”指天,“μL”指微升，“ml”指毫升，“L“指升，“bp”指碱基对，“mM”指毫摩尔，“μM”指微摩尔。
实施例1：以合成K205R基因质粒DNA为模板进行RPA反应
1)K205R基因保守区的选择和基因克隆：对来自GeneBank上已提交并认证过的24种ASFV病毒株(Table1)的全基因组序列进行同源性比较，寻找出一段K205R基因的保守区作为RPA扩增的序列(196bp)，分析24种ASFV在该基因区段上可变碱基的位点(表2)；直接合成K205R基因片段(SEQ ID NO.11)，克隆至pUC-57载体(GenScriptCat.No.SD1176)，合成质粒pUC-57-K205R。
2)RPA引物的设计、合成：根据RPA引物的设计要求，RPA引物最多包含两个可变碱基位点，设计的上游引物RPA-F(SEQ ID NO.35)和下游引物RPA-R(SEQ ID NO.36)序列见Table3；
3)RPA反应以及琼脂糖凝胶电泳：将上述设计合成的引物稀释到10uM终浓度，在含有RPA反应酶冻干粉的反应体系中加入上游引物RPA-F(SEQ ID NO.35)和下游引物RPA-R(SEQ ID NO.36)各2.4ul，RPA buffer(RPA试剂盒内提供)29.5ul，10 4(用1E4表示)和10 8(用1E8表示)不同拷贝量的pUC-57-K205R质粒DNA，3.5μl 280 mMMgAc离子， 加ddH 2O补齐至50ul，37℃反应15min。吸取5ul反应产物，在2％的琼脂糖凝胶中进行电泳检测，凝胶成像结果如图1所示，结果显示该对引物可以对K205R基因质粒DNA有效的进行RPA扩增，扩增产物长度209bp；
实施例2：针对K205R基因的Cas12a-crRNA有效性的验证
本实施例2中，T1-crRNA1与Cas12a蛋白形成复合体后对K205R基因进行特异性切割和ssDNA荧光探针进行非特异性切割的荧光检测。
1)crRNA的制备：T1-crRNA1由苏州金唯智生物科技有限公司合成，T1-crRNA1的RNA序列(SEQ ID NO.37)见表3,合成后的RNA稀释至20uM,-20℃保存备用。
2)Cas12a-crRNA结合反应：在10ul缓冲液中(40mM Tris-HCl；60mM NaCl；6 mM MgCl 2)加入浓度为40uM的LbCas12a蛋白10ul(SEQIDNo:1，购自Integrated DNA Technologies)和浓度为20uM的crRNA 20ul(T1-crRNA1)，配制成Cas12a-crRNA终浓度均为10uM的体系，室温孵育20min；
3)检测体系的配制：在每个检测体系中加入5ul步骤2)中的Cas12a-crRNA复合体，加入1ul 100nM的实施例1制得的pUC-57-K205R质粒DNA，44ul的缓冲液(40mM Tris-HCl；60mM NaCl；6 mM MgCl2)，混匀后加入到含有单链DNA荧光探针的DNaseAlert TM Substrate(Integrated DNA Technologies)中，吹打混匀；
4)荧光信号的检测：将反应液快速转移至96孔半孔酶标仪板在酶标仪中进行信号检测：在Ex/Em＝535/560nM波长条件下进行连续动力学荧光信号检测，每隔2min收集荧光信号一次，连续监测1-2h；
5)荧光检测结果分析：结果如图2所示。
使用T2-crRNA1的RNA序列(SEQ ID NO.38)来代替T1-crRNA1，在上述相同的实验条件下操作。图2结果显示，对于100nM浓度的DNA，T1-crRNA1和T2-crRNA1组荧光值的信号在15分钟左右可以表现出显著的增强，表明实现了有效率的切割。
实施例3.不同初始量的K205R基因进行Cas12a基本检测
1)Cas12a-crRNA结合反应：在10ul缓冲液中(40mM Tris-HCl；60mM NaCl；6 mM MgCl 2)加入10ul LbCas12a蛋白(购自Integrated DNA Technologies)(40uM)和20ul T1-crRNA1(20uM)，配置成Cas12a-crRNA终浓度均为10uM的体系，室温孵育20min；
2)检测体系的配制：在检测体系中加入5ul步骤1)中的Cas12a-crRNA复合体，加入1ul不同载量的pUC-57-K205R质粒DNA(分别为10 7、10 8、10 9、10 10拷贝，分别用E7、E8、E9、E10表示)，44ul的缓冲液(40mM Tris-HCl；60mM NaCl；6 mM MgCl2)，混匀后加入到含有单链DNA荧光探针的DNaseAlert TM Substrate(购自Integrated DNA Technologies)中，吹打混匀；
3)荧光信号的检测：将反应液快速转移至96孔半孔酶标仪板在酶标仪中进行信号检测：在Ex/Em＝535/560nM波长条件下进行连续动力学荧光信号检测，每隔2min收集荧光信号一次，连续监测1-2h；
4)荧光检测结果分析：结果如图3所示，对于10 10拷贝的DNA，荧光值的信号在15分钟左右可以表现出显著的差异；对于10 9拷贝的DNA，荧光值的信号在40分钟左右可以表现出显著的差异；对于更低载量的DNA，荧光值的信号与NC组(未加靶标DNA的阴性对照组)没有显著差异；
表3
Figure PCTCN2020093456-appb-000025
实施例4.基于Cas12a和RPA联合的非洲猪瘟病毒检测方法，以合成的非洲猪瘟病毒K205R基因部分保守区序列的质粒DNA作为靶标进行检测
1)RPA预扩增反应：在含有RPA反应酶冻干粉的反应体系中加入上游引物RPA-F(SEQ ID NO.35)和下游引物RPA-R(SEQ ID NO.36)各2.4ul，RPA buffer(RPA试剂盒内提供，购自TwistDx)29.5ul，加入1ul 10倍浓度梯度稀释的不同载量的pUC-57-K205R质粒DNA(10、10 2、10 4、10 8拷贝每微升：图中分别用E1、E2、E4、E8表示)，3.5μl 280 mMMgAc离子，加ddH 2O补齐至50ul，37℃反应15min。
2)Cas12a-crRNA结合反应：在10ul缓冲液中(40mM Tris-HCl；60mM NaCl；6 mM MgCl2)加入10ul LbCas12a蛋白(Integrated DNA Technologies)(40uM)和20ul T1-crRNA1(20uM)，配置成Cas12a-crRNA终浓度均为10μM的体系，室温孵育20min.
3)Cas12a荧光检测反应体系：
取20ul上述RPA反应产物加入到含有单链DNA荧光探针的DNaseAlert TM Substrate(Integrated DNA Technologies)中，同时加入5ul步骤2中孵育好的Cas12a-crRNA，控制500nM终浓度的Cas12a-crRNA，1ul RNase抑制剂(TaKaRa)，加24ul缓冲液(40mM Tris-HCl；60mM NaCl；6 mM MgCl 2)补齐至50ul；将反应液快速转移至96孔半孔白色底透微孔板准备进行酶标仪上机进行荧光信号的检测：将反应液快速转移至96孔半孔酶标仪板在酶标仪中进行信号检测：在Ex/Em＝535/560nM波长条件下进行连续动力学荧光信号检测，每隔2min收集荧光信号一次，连续监测1-2h；
4)检测灵敏度结果分析：结果如图4-1显示，荧光值随靶标的浓度和反应时间的增加而增加；本方法可以检测低至10拷贝/ul非洲猪瘟病毒pUC-57-K205R质粒DNA，在30min检测时间时，与无模板阴性对照组荧光数值存在显著差异；如图4-2对于10拷贝/ul非洲猪瘟病毒pUC-57-K205R质粒DNA，检测反应20min，荧光数值即可展现出显著差异；本方法流程简单快速，10拷贝的检测实验周期时长1h左右，高载量的检测时长低至30-45min左右。

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  1. 一种用于检测非洲猪瘟病毒的核酸检测体系，包含：
    a)Cas12蛋白；b)一个或多个crRNA，其中crRNA能与对应的非洲猪瘟病毒的基因片段结合；和c)单链DNA荧光探针。
  2. 权利要求1所述的检测体系，其中所述的非洲猪瘟病毒的基因为K205R、CP530R、CP204L和P72，优选为K205R基因。
  3. 权利要求1或2所述的检测体系，其中所述的Cas12蛋白选自Cas12a，Cas12b或Cas12c。
  4. 权利要求3所述的检测体系，其中所述的Cas12a蛋白选自FnCas12a、AsCas12a、LbCas12a、Lb5Cas12a、HkCas12a、OsCas12a、TsCas12a、BbCas12a、BoCas12a或Lb4Cas12a。
  5. 权利要求4所述的检测体系，其中所述的Cas12a蛋白具有SEQIDNo：1所示的序列；或与SEQIDNo：1所示的序列具有至少80％同一性的序列；或与SEQIDNo：1所示的序列具有一个或几个氨基酸的取代、缺失和***的Cas12a蛋白的变体。
  6. 权利要求5所述的检测体系，其中所述的Cas12a蛋白与SEQIDNo：1所示的序列具有至少83％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％同一性的序列。
  7. 前述任一项权利要求所述的检测体系，其中所述的非洲猪瘟病毒的基因片段具有至少15bp的长度，优选具有至少20bp的长度。
  8. 前述任一项权利要求所述的检测体系，其中所述的crRNA的指导序列具有15nt-28nt的长度。
  9. 前述任一项权利要求所述的检测体系，其中所述的crRNA具有29-100nt的长度。
  10. 权利要求9所述的检测体系，其中所述的crRNA具有40-50nt的长度。
  11. 前述任一项权利要求所述的检测体系，其中多个单链crRNA结合同一个非洲猪瘟病毒的基因片段。
  12. 前述任一项权利要求所述的检测体系，其中多个单链crRNA结合不同的非洲猪瘟病毒的基因片段。
  13. 前述任一项权利要求所述的检测体系，其中所述的单链DNA荧光探针的5’端和3’端分别标记荧光基团和猝灭基团。
  14. 前述任一项权利要求所述的检测体系，还进一步包含非洲猪瘟病毒的核酸的扩增体系。
  15. 权利要求14所述的检测体系，其中所述的扩增体系为重组酶聚合酶扩增体系。
  16. 权利要求14所述的检测体系，其中所述的扩增体系为PCR扩增体系。
  17. 前述任一项权利要求所述的检测体系，其中所述的非洲猪瘟病毒的核酸来自于猪的组织，该组织优选是全血、猪血浆、猪血清、猪尿液、猪唾液或猪口腔粘膜。
  18. 一种检测非洲猪瘟病毒的核酸检测方法，包含：
    a.将核酸样品与权利要求1-17任一项所述的检测体系在20℃-42℃孵育，检测荧光强度的变化；
    b.所述的核酸样品是从非洲猪瘟病毒中提取的样品DNA，或者是从非洲猪瘟病毒中提取的样品DNA经过扩增而得到的靶标DNA。
  19. 权利要求17或18述的检测方法，其中，所述的DNA扩增为重组酶聚合酶扩增或者PCR扩增。
  20. 权利要求18或19所述的检测体系，其中所述的核酸样品来自于猪的组织，该组织优选是全血、猪血浆、猪血清、猪尿液、猪唾液或猪口腔粘膜。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112795673A (zh) * 2021-02-09 2021-05-14 上海市质量监督检验技术研究院 一种食品中克罗诺杆菌属的crispr检测方法及其试剂盒
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CN113073146A (zh) * 2021-03-30 2021-07-06 铭基食品有限公司 Bax全自动病原微生物快速检测***在非洲猪瘟病毒检测中的应用
CN113969320A (zh) * 2021-09-26 2022-01-25 华南农业大学 一种用于鉴别猪源性成分的rpa引物及检测体系和方法
CN114015791A (zh) * 2021-09-13 2022-02-08 东莞理工学院 基于AIE-CRISPR/cas12a对环境中病原微生物的可视化检测方法
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* Cited by examiner, † Cited by third party
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101463396A (zh) * 2009-01-06 2009-06-24 天津出入境检验检疫局动植物与食品检测中心 非洲猪瘟病毒荧光定量pcr检测试剂及其制备方法和用途
CN101921878A (zh) * 2010-09-21 2010-12-22 扬州大学 一种非洲猪瘟病毒核酸扩增引物、检测方法和试剂盒
WO2012079016A1 (en) * 2010-12-10 2012-06-14 Brandeis University Compositions and methods for the detection and analysis of african swine fever virus
WO2013055789A1 (en) * 2011-10-14 2013-04-18 Accugenomics, Inc. Nucleic acid amplification and use thereof
CN110129485A (zh) * 2019-06-10 2019-08-16 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 一种快速鉴别非洲猪瘟病毒的检测试剂盒及其检测方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108707621B (zh) * 2018-04-26 2021-02-12 中国农业科学院作物科学研究所 一种CRISPR/Cpf1***介导的以RNA转录本为修复模板的同源重组方法
CN110453011B (zh) * 2019-07-19 2021-09-07 中山大学 一种基于CRISPR/Cas12a快速精准检测非洲猪瘟病毒的方法及应用
CN110894557A (zh) * 2019-09-20 2020-03-20 武汉大学 基于CRISPR方式检测非洲猪瘟病毒的crRNA及试剂盒
CN111254223A (zh) * 2020-03-21 2020-06-09 上海海关动植物与食品检验检疫技术中心 一种用于检测非洲猪瘟病毒核酸的反应体系、试剂盒及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101463396A (zh) * 2009-01-06 2009-06-24 天津出入境检验检疫局动植物与食品检测中心 非洲猪瘟病毒荧光定量pcr检测试剂及其制备方法和用途
CN101921878A (zh) * 2010-09-21 2010-12-22 扬州大学 一种非洲猪瘟病毒核酸扩增引物、检测方法和试剂盒
WO2012079016A1 (en) * 2010-12-10 2012-06-14 Brandeis University Compositions and methods for the detection and analysis of african swine fever virus
WO2013055789A1 (en) * 2011-10-14 2013-04-18 Accugenomics, Inc. Nucleic acid amplification and use thereof
CN110129485A (zh) * 2019-06-10 2019-08-16 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 一种快速鉴别非洲猪瘟病毒的检测试剂盒及其检测方法

Cited By (14)

* Cited by examiner, † Cited by third party
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CN112795673A (zh) * 2021-02-09 2021-05-14 上海市质量监督检验技术研究院 一种食品中克罗诺杆菌属的crispr检测方法及其试剂盒
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CN114381553B (zh) * 2022-01-27 2023-07-04 云南大学 非洲猪瘟病毒检测用生物材料、试剂盒和非诊断目的的非洲猪瘟病毒的检测方法
CN116064963B (zh) * 2022-11-25 2023-09-19 深圳海关动植物检验检疫技术中心 一种基于CRISPR-Cas12a的非洲马瘟病毒可视化快速检测方法及其试剂盒
CN116064963A (zh) * 2022-11-25 2023-05-05 深圳海关动植物检验检疫技术中心 一种基于CRISPR-Cas12a的非洲马瘟病毒可视化快速检测方法及其试剂盒
WO2024125238A1 (zh) * 2022-12-15 2024-06-20 中国科学院深圳先进技术研究院 病毒核酸检测试剂、试剂盒、制备方法及其应用
CN116287448A (zh) * 2023-02-06 2023-06-23 河北农业大学 一种用于非洲猪瘟病毒检测的raa引物对、探针及其应用

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