CN113046480A

CN113046480A - Method for rapidly detecting African swine fever virus based on CRISPR-Cas13a protein

Info

Publication number: CN113046480A
Application number: CN202110311002.2A
Authority: CN
Inventors: 曾建红; 张小兰
Original assignee: Leshang Biotechnology Wuxi Co ltd
Current assignee: Leshang Biotechnology Wuxi Co ltd
Priority date: 2021-03-23
Filing date: 2021-03-23
Publication date: 2021-06-29

Abstract

The invention relates to the technical field of molecular biology, in particular to a method for rapidly detecting African swine fever virus based on CRISPR-Cas13a protein, which comprises the steps of preparing Cas13a protein, designing and synthesizing a primer suitable for RPA amplification, designing and synthesizing crRNA suitable for Cas13a protein, designing and synthesizing an RNA reporter molecule, designing and synthesizing positive plasmid, preparing an amplification detection system and detecting. The method can quickly and accurately identify the African swine fever virus DNA, is simple and convenient to operate, does not need to realize amplification detection by a variable temperature instrument like PCR, and can finish detection only by carrying out isothermal amplification at 37 ℃.

Description

Method for rapidly detecting African swine fever virus based on CRISPR-Cas13a protein

Technical Field

The invention relates to the technical field of molecular biology, in particular to a method for rapidly detecting African swine fever virus based on CRISPR-Cas13a protein.

Background

African Swine Fever (ASF) is an infectious disease in pigs caused by African Swine Fever Virus (ASFV). The African swine fever has the advantages of fast spread, high lethality rate and great harm to the swine industry, is listed as an animal epidemic disease needing to be reported by the world animal health Organization (OIE), and is listed as an animal disease in China. To date, African swine fever has been outbreak in several dozen countries, including Africa, Europe, and America. The discovery situation of the African swine fever in the Chinese epidemic situation is very severe, and causes great risk to the breeding industry in China. At present, the African swine fever virus has no effective vaccine and medicament, the prevention and treatment difficulty is high, and the only effective measure is to carry out killing. In addition, the African swine fever virus has extremely strong viability and various propagation ways, and easily causes spreading and expansion of epidemic situations. Therefore, it is necessary to establish a simple, rapid and accurate method for detecting African swine fever virus.

At present, the detection method of African swine fever virus mainly comprises immunodetection and molecular detection. The immunoassay, namely the enzyme-linked immunosorbent assay of the antibody and the antigen, which is called ELISA assay for short, is applied to the detection of serum antibody, has the characteristics of convenient operation, better specificity and high sensitivity, is suitable for the detection of mass samples, and the world animal health organization takes the ELISA as the first choice serological method for diagnosing ASF. Molecular detection, mainly a PCR method, is the most common laboratory detection method for African swine fever virus at present.

The serological method for detecting the antibody can understand the virus infection and the disease occurrence and development processes, however, the antibody can only appear after the virus infection reaches a certain period, and even some sick pigs still do not appear the antibody after the death of the sick pigs because the disease process develops rapidly. Therefore, antibody detection has limitations as a means of rapidly controlling the outbreak of ASFV. The PCR detection has the advantages of high flux, intuitive result, high sensitivity, strong specificity, good repeatability and the like, and becomes an important method for detecting the pathogen of the African swine fever. However, the PCR method requires expensive instruments, has high requirements on the experimental field and personnel, and is difficult to be suitable for large-scale field screening.

Therefore, a new technical solution is urgently needed to solve the above problems.

Disclosure of Invention

The invention aims to overcome the problems in the prior art, and provides a method for rapidly detecting the African swine fever virus based on CRISPR-Cas13a protein, which can conveniently, rapidly and accurately identify the DNA of the African swine fever virus, is simple and convenient to operate, does not need a temperature-variable instrument to realize amplification detection like PCR, and can finish detection only by carrying out isothermal amplification at 37 ℃.

The above purpose is realized by the following technical scheme:

a method for rapidly detecting African swine fever virus based on CRISPR-Cas13a protein comprises the following steps:

step (1) preparing a Cas13a protein;

designing and synthesizing primers suitable for RPA amplification;

designing and synthesizing crRNA suitable for the Casl3a protein in the step (3);

designing and synthesizing an RNA (ribonucleic acid) reporter molecule;

designing and synthesizing positive plasmids;

preparing positive plasmids;

step (7), preparing an amplification detection system;

and (8) detecting.

Further, the step (1) specifically comprises the following steps:

searching a Cas13a gene sequence coded by Leptotrichia wadei on NCBI, wherein the nucleotide sequence of the Cas13a gene is shown as SEQ ID No.5, and the amino acid sequence is shown as SEQ ID No.6, artificially synthesizing to obtain a full-length Cas13a gene fragment, cloning the full-length Cas13a gene fragment to a T vector, carrying out double enzyme digestion by NdeI and SacI, cloning the fragment to a position between NdeI and SacI enzyme digestion sites of a pET-28a expression vector, and constructing an expression vector pET-28a-Cas13 a;

transforming the expression vector pET-28a-Cas13a constructed in the step (a) into escherichia coli BL21(DE3), coating the escherichia coli BL21 on a Kan plate containing 30 mu g/ml, culturing for 12h-16h at 37 ℃, selecting a monoclonal strain, adding 1L of LB liquid culture medium of Kan with the final concentration of 30 mu g/ml for culturing for 4h, adding IPTG with the final concentration of 0.7mM for induction, culturing at 30 ℃ overnight, collecting a bacterial liquid, and centrifuging at 5000rpm for 30min to obtain thalli;

and (c) carrying out ultrasonic crushing on the thalli obtained in the step (b) by using a buffer solution, taking a supernatant, centrifuging for 30min at 10000rpm, washing the supernatant to a baseline level by using the buffer solution after purification, eluting the protein by using the buffer solution containing 250mM of imidazole, desalting the eluted protein by using a sephadex G25 chromatographic column to obtain the protein, and storing at-70 ℃ to obtain the purified Cas13a protein.

Further, the step (2) is specifically: designing an upstream primer and a downstream primer for specifically detecting the African swine fever virus according to a conserved African swine fever virus VP72 gene, performing blast on the designed upstream primer and downstream primer in NCBI, and displaying the sequence of the African swine fever virus as a specific sequence;

the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1;

the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2;

the sequence of the African swine fever virus VP72 gene is shown in SEQ ID NO. 4.

Further, the step (3) is specifically: according to the characteristics of the Cas13a protein, crRNA for specifically recognizing African swine fever virus is directly designed in the sequence of the amplified gene fragment, blast is carried out in NCBI, and the sequence is shown to be a specific African swine fever virus sequence; the nucleic acid sequence of the crRNA is shown as SEQ ID NO. 3.

Further, the step (4) is specifically as follows: designing an RNA reporter molecule according to the characteristics of Cas13a protein, wherein the sequence of the RNA reporter molecule is a fluorescent reporter group-TUUUUC-fluorescent quencher group, and the fluorescent reporter group is FAM, HEX, TET, JOE or VIC; the fluorescence quenching group is BHQ1, BHQ2 or BHQ 3.

Furthermore, the sequence of the RNA reporter molecule is FAM-TUUUUUC-BHQ1, the 5 'end is modified with FMA fluorescent group, and the 3' end is modified with BHQ1 quenching group.

Further, the step (5) is specifically: the nucleotide sequence of the African swine fever virus VP72 gene is cloned into pUc57 plasmid, the cloning site is SamI, the resistance is ampicillin resistance, and the cloning strain is DH5 d.

Further, the step (6) is specifically: inoculating the positive plasmid obtained in step (5) into LB medium containing 100. mu.g/ml ampicillin in an amount of 1%, and culturing overnight at 37 ℃ and 120rpm in a shaker; and (3) sucking bacterial liquid, extracting plasmids by adopting a Tiangen plasmid extraction kit, measuring the concentration by using Nanodrop 2000, calculating the copy number, and performing serial gradient dilution for subsequent use.

Further, the amplification detection system in the step (7) comprises the following components and dosage:

further, the step (8) is specifically: and (3) preparing a recombinant plasmid working standard substance according to the positive plasmid prepared in the step (6), adding the working standard substance into the amplification detection system and the RPA reaction unit in the step (7), testing the reaction by a normal-temperature isothermal amplification fluorescence detector, and observing the result.

Advantageous effects

The method for rapidly detecting the African swine fever virus based on the CRISPR-Cas13a protein, provided by the invention, not only overcomes the defects that a PCR method needs expensive instruments, has higher requirements on an experimental site and personnel and is difficult to be suitable for large-scale field screening, but also can conveniently, rapidly and accurately identify the DNA of the African swine fever virus, is simple and convenient to operate, does not need a temperature-variable instrument like PCR to realize amplification detection, and can finish detection only by carrying out isothermal amplification at 37 ℃.

Drawings

FIG. 1 is a schematic diagram of a method for rapidly detecting African swine fever virus based on CRISPR-Cas13a protein according to the invention;

FIG. 2 is a result of detecting the sensitivity of the African swine fever virus plasmid DNA at different concentrations in the method for rapidly detecting the African swine fever virus based on the CRISPR-Cas13a protein;

FIG. 3 shows the result of repetitive detection of African swine fever virus plasmid DNA in the method for rapidly detecting African swine fever virus based on CRISPR-Cas13a protein.

Detailed Description

The invention is explained in more detail below with reference to the figures and examples.

step 1: preparing a Cas13a protein;

step 2: designing and synthesizing primers suitable for RPA amplification;

and step 3: designing and synthesizing crRNA suitable for Cas13a protein;

and 4, step 4: designing and synthesizing an RNA reporter molecule;

and 5: designing and synthesizing positive plasmids;

step 6: preparing positive plasmids;

the method comprises the following steps: 7: preparing an amplification detection system;

the method comprises the following steps: 8: and (6) detecting.

The specific operation of each step is as follows:

the step (1) comprises the following steps:

step (a) construction of an expression vector

Searching a sequence of a Leptotrichia wadei encoding Cas13a gene on NCBI, wherein the sequence number of GenBank is WP-021746774.1, the nucleotide sequence of the Cas13a gene is shown as SEQ ID No.5, and the amino acid sequence of a protein encoded by the gene is shown as SEQ ID No.6, artificially synthesizing to obtain a full-length Cas13a gene fragment (the nucleotide sequence of which is shown as SEQ ID No. 5), cloning the full-length Cas13a gene fragment to a T vector, carrying out double enzyme digestion by NdeI and SacI, cloning the full-length Cas13 gene fragment to a position between NdeI and SacI enzyme digestion sites of a pET-28a expression vector, and constructing an expression vector pET-28a-Cas13 a;

step (b) constructing recombinant engineering bacteria and fermenting

Transforming the expression vector pET-28a-Cas13a constructed in the step (a) into escherichia coli BL21(DE3), coating the escherichia coli BL21 on a Kan plate containing 30 mu g/ml, culturing for 12h-16h at 37 ℃, selecting a monoclonal strain, adding 1L of LB liquid culture medium of Kan with the final concentration of 30 mu g/ml for culturing for 4h, adding IPTG with the final concentration of 0.7mM for induction, culturing overnight at 30 ℃, collecting a bacterial liquid, and centrifuging at 5000rpm for 30min to obtain thalli;

step (c) Cas13a protein purification

And (c) carrying out ultrasonic disruption on the thalli obtained in the step (b) by using a buffer solution, taking a supernatant, centrifuging for 30min at 10000rpm, purifying by using Ni SwpharoseTM 6Fast Flow (a purchased company and a product number: GE, 11-0008-87AF), washing to a baseline level by using the buffer solution, eluting the protein by using the buffer solution with the imidazole concentration of 250mM, desalting the eluted protein by using a sephadex G25(GE, 17-0033-01) chromatographic column to obtain the protein, and storing at-70 ℃ to obtain the purified Cas13a protein.

The step (2) designs and synthesizes primers suitable for RPA amplification, and specifically comprises the following steps:

according to the design principle of the RPA kit, primers are designed, the length of the primers is required to be about 30nt, and the amplified fragment is within 500 bp. Designing an upstream primer and a downstream primer for specifically detecting the African swine fever virus according to a conserved African swine fever virus VP72 gene (the sequence of the African swine fever virus VP72 gene is shown as SEQ ID N0.4), and performing blast on the designed upstream primer and the designed downstream primer in NCBI to show that the sequence is a specific African swine fever virus sequence;

in the scheme, the nucleotide sequence of the upstream primer is shown as SEQ ID N0.1; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2.

The step (3) designs and synthesizes crRNA suitable for the Cas13a protein, and specifically comprises the following steps:

according to the characteristics of the Cas13a protein, a crRNA (the nucleic acid sequence of which is shown as SEQ ID NO. 3) for specifically recognizing African swine fever virus is directly designed in the sequence of the amplified gene fragment, and blast is carried out in NCBI to show that the sequence is a specific African swine fever virus sequence.

The step (4) of designing and synthesizing the RNA reporter molecule specifically comprises the following steps:

designing an RNA reporter molecule according to the characteristics of Cas13a protein, wherein the sequence of the RNA reporter molecule is a fluorescent reporter group-TUUUUC-fluorescent quencher group, and the fluorescent reporter group is FAM, HEX, TET, JOE or VIC; the fluorescence quenching group is BHQ1, BHQ2 or BHQ 3.

Preferably, the sequence of the RNA reporter molecule is FAM-TUUUUC-BHQ 1, the 5 'end is modified with FMA fluorescent group, and the 3' end is modified with BHQ1 quenching group.

In the step (3) and the step (4), the Cas13a protein is preferably LwCas13 a.

The step (5) designs and synthesizes positive plasmids, which specifically comprise the following steps:

the nucleotide sequence of the African swine fever virus VP72 gene (shown as SEQ ID NO. 4) is cloned into pUc57 plasmid, the cloning site is SamI, the resistance is ampicillin resistance, and the cloning strain is DH5 d.

The step (6) of preparing positive plasmids specifically comprises the following steps:

inoculating the DH5 alpha bacterial liquid containing the recombinant plasmid obtained in the step (5) into LB culture medium containing 100 mu g/ml ampicillin in an inoculation amount of 1%, and culturing overnight at 37 ℃ and 120rpm in a shaking table; and (3) sucking bacterial liquid, extracting plasmids by adopting a Tiangen plasmid extraction kit according to the operation steps of the instruction, measuring the concentration by using Nanodrop 2000, calculating the copy number, and performing serial gradient dilution for subsequent use.

The amplification detection system in the step (7) comprises the following components and dosage:

according to the required detection quantity, a corresponding reaction system can be prepared, and all the components are sequentially added for reaction.

The detection in the step (8) is specifically as follows:

and (3) preparing a recombinant plasmid working standard substance according to the positive plasmid prepared in the step (6), adding the working standard substance into the amplification detection system and the RPA reaction unit in the step (7), testing the reaction by a normal-temperature isothermal amplification fluorescence detector, and observing the result. The normal-temperature isothermal amplification fluorescence detector is an instrument capable of detecting FAM fluorescence, the reaction temperature is set to 37 ℃, the reaction time is set to 60 minutes, and the fluorescence is observed in real time.

The following examples are further described

Example one

Sensitivity test

(1) Synthesis of Positive plasmids

The nucleic acid sequence (shown as SEQ ID NO. 4) was cloned into pUc57 plasmid (Shanghai's work was entrusted), the cloning site was SamI, and the copy number was calculated by measuring the concentration with Nanodrop 2000.

(2) Preparing recombinant plasmid working standard products which are respectively as follows:

working standard 1, containing 1.0 × 10⁶Plasmid of DNA fragment of Copies/mu L African swine fever virus.

Working standard 2, containing 1.0 × 10⁵Plasmid of DNA fragment of Copies/mu L African swine fever virus.

Working standard 3, containing 1.0 × 10⁴Plasmid of DNA fragment of Copies/mu L African swine fever virus.

Working standard 4, containing 1.0 × 10³Plasmid of DNA fragment of Copies/mu L African swine fever virus.

Working standard 5, containing 1.0 × 10²Plasmid of DNA fragment of Copies/mu L African swine fever virus.

Working standard 6, containing 1.0 × 10¹Plasmid of DNA fragment of Copies/mu L African swine fever virus.

(3) Preparing a reaction system

Preparing reaction solution (prepared according to 8 reactions) according to the amplification system in step 7, mixing uniformly in a 1.5ml EP tube, sucking 46. mu.L of the reaction solution, and adding the mixture into 7 RPA reaction units respectively (R) ((R))

Basic, TwistdX, the kit can realize DNA amplification. Containing all enzymes and reagents required by DNA amplification, and a user only needs to provide primers and templates), dissolving the freeze-dried powder, respectively adding 1 mu L of negative quality control materials, 1 mu L of standard works 6, 1 mu L of standard works 5, 1 mu L of standard works 4, 1 mu L of standard works 3, 1 mu L of standard works 2 and 1 mu L of standard works 1 as templates into the 7 prepared reaction reagent test tubes, adding 3 mu L of 280mM magnesium acetate onto the tube covers, and fully mixing each reaction tube after sample addition, wherein the total volume of each reaction tube is 50 mu L.

The uniformly mixed 7 reaction tubes were placed in a normal temperature isothermal amplification fluorescence detector (T8 portable normal temperature isothermal amplification fluorescence detector, twist dx), the reaction temperature was set at 37 ℃, and the reaction time was 60 minutes.

The detection results are shown in figure 2;

the results show that amplification signals are generated within 60 minutes, the detection sensitivity can reach 1.0 multiplied by 101 Copies/reaction, namely 10Copies exist in each reaction tube, the detection can be realized within 60 minutes, and the quick and sensitive detection result is realized.

Example two

Repeatability test

The materials, the amounts and the positive quality control materials are the same as those in the first embodiment.

Using working standard 5 containing 1.0 × 10²Plasmid of DNA fragment of Copies/ul African virus.

Preparing reaction solution (prepared according to 9 reactions) according to the amplification system in the step 7, mixing the reaction solution in a 1.5ml centrifuge tube uniformly, sucking 6 mul, and adding the mixture into 8 RPA reaction units respectively (R) ((R))

Basic, twist Dx), adding 1 μ L of standard 5 into 8, adding 1 μ L of negative quality control material into 1, adding 3 μ L of 280mM magnesium acetate onto the tube cover, mixing each reaction tube, and making the total volume of each reaction tube be 50 μ L. The uniformly mixed 9 reaction tubes were placed in a normal temperature isothermal amplification fluorescence detector (T8 portable normal temperature isothermal amplification fluorescence detector, twist dx), the reaction temperature was set at 39 ℃, and the reaction time was 60 minutes.

The detection results are shown in FIG. 3;

the result shows that the sensitivity of each reaction tube can reach 1.0 multiplied by 103Copies, and the repeatability is good.

The above description is for the purpose of illustrating embodiments of the invention and is not intended to limit the invention, and it will be understood by those skilled in the art that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

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Glu Asn Lys Gln Asn Glu Val Lys Glu Asn Leu Lys Met Phe Tyr Ser

435 440 445

Tyr Asp Phe Asn Met Asp Asn Lys Asn Glu Ile Glu Asp Phe Phe Ala

450 455 460

Asn Ile Asp Glu Ala Ile Ser Ser Ile Arg His Gly Ile Val His Phe

465 470 475 480

Asn Leu Glu Leu Glu Gly Lys Asp Ile Phe Ala Phe Lys Asn Ile Ala

485 490 495

Pro Ser Glu Ile Ser Lys Lys Met Phe Gln Asn Glu Ile Asn Glu Lys

500 505 510

Lys Leu Lys Leu Lys Ile Phe Lys Gln Leu Asn Ser Ala Asn Val Phe

515 520 525

Asn Tyr Tyr Glu Lys Asp Val Ile Ile Lys Tyr Leu Lys Asn Thr Lys

530 535 540

Phe Asn Phe Val Asn Lys Asn Ile Pro Phe Val Pro Ser Phe Thr Lys

545 550 555 560

Leu Tyr Asn Lys Ile Glu Asp Leu Arg Asn Thr Leu Lys Phe Phe Trp

565 570 575

Ser Val Pro Lys Asp Lys Glu Glu Lys Asp Ala Gln Ile Tyr Leu Leu

580 585 590

Lys Asn Ile Tyr Tyr Gly Glu Phe Leu Asn Lys Phe Val Lys Asn Ser

595 600 605

Lys Val Phe Phe Lys Ile Thr Asn Glu Val Ile Lys Ile Asn Lys Gln

610 615 620

Arg Asn Gln Lys Thr Gly His Tyr Lys Tyr Gln Lys Phe Glu Asn Ile

625 630 635 640

Glu Lys Thr Val Pro Val Glu Tyr Leu Ala Ile Ile Gln Ser Arg Glu

645 650 655

Met Ile Asn Asn Gln Asp Lys Glu Glu Lys Asn Thr Tyr Ile Asp Phe

660 665 670

Ile Gln Gln Ile Phe Leu Lys Gly Phe Ile Asp Tyr Leu Asn Lys Asn

675 680 685

Asn Leu Lys Tyr Ile Glu Ser Asn Asn Asn Asn Asp Asn Asn Asp Ile

690 695 700

Phe Ser Lys Ile Lys Ile Lys Lys Asp Asn Lys Glu Lys Tyr Asp Lys

705 710 715 720

Ile Leu Lys Asn Tyr Glu Lys His Asn Arg Asn Lys Glu Ile Pro His

725 730 735

Glu Ile Asn Glu Phe Val Arg Glu Ile Lys Leu Gly Lys Ile Leu Lys

740 745 750

Tyr Thr Glu Asn Leu Asn Met Phe Tyr Leu Ile Leu Lys Leu Leu Asn

755 760 765

His Lys Glu Leu Thr Asn Leu Lys Gly Ser Leu Glu Lys Tyr Gln Ser

770 775 780

Ala Asn Lys Glu Glu Thr Phe Ser Asp Glu Leu Glu Leu Ile Asn Leu

785 790 795 800

Leu Asn Leu Asp Asn Asn Arg Val Thr Glu Asp Phe Glu Leu Glu Ala

805 810 815

Asn Glu Ile Gly Lys Phe Leu Asp Phe Asn Glu Asn Lys Ile Lys Asp

820 825 830

Arg Lys Glu Leu Lys Lys Phe Asp Thr Asn Lys Ile Tyr Phe Asp Gly

835 840 845

Glu Asn Ile Ile Lys His Arg Ala Phe Tyr Asn Ile Lys Lys Tyr Gly

850 855 860

Met Leu Asn Leu Leu Glu Lys Ile Ala Asp Lys Ala Lys Tyr Lys Ile

865 870 875 880

Ser Leu Lys Glu Leu Lys Glu Tyr Ser Asn Lys Lys Asn Glu Ile Glu

885 890 895

Lys Asn Tyr Thr Met Gln Gln Asn Leu His Arg Lys Tyr Ala Arg Pro

900 905 910

Lys Lys Asp Glu Lys Phe Asn Asp Glu Asp Tyr Lys Glu Tyr Glu Lys

915 920 925

Ala Ile Gly Asn Ile Gln Lys Tyr Thr His Leu Lys Asn Lys Val Glu

930 935 940

Phe Asn Glu Leu Asn Leu Leu Gln Gly Leu Leu Leu Lys Ile Leu His

945 950 955 960

Arg Leu Val Gly Tyr Thr Ser Ile Trp Glu Arg Asp Leu Arg Phe Arg

965 970 975

Leu Lys Gly Glu Phe Pro Glu Asn His Tyr Ile Glu Glu Ile Phe Asn

980 985 990

Phe Asp Asn Ser Lys Asn Val Lys Tyr Lys Ser Gly Gln Ile Val Glu

995 1000 1005

Lys Tyr Ile Asn Phe Tyr Lys Glu Leu Tyr Lys Asp Asn Val Glu Lys

1010 1015 1020

Arg Ser Ile Tyr Ser Asp Lys Lys Val Lys Lys Leu Lys Gln Glu Lys

1025 1030 1035 1040

Lys Asp Leu Tyr Ile Arg Asn Tyr Ile Ala His Phe Asn Tyr Ile Pro

1045 1050 1055

His Ala Glu Ile Ser Leu Leu Glu Val Leu Glu Asn Leu Arg Lys Leu

1060 1065 1070

Leu Ser Tyr Asp Arg Lys Leu Lys Asn Ala Ile Met Lys Ser Ile Val

1075 1080 1085

Asp Ile Leu Lys Glu Tyr Gly Phe Val Ala Thr Phe Lys Ile Gly Ala

1090 1095 1100

Asp Lys Lys Ile Glu Ile Gln Thr Leu Glu Ser Glu Lys Ile Val His

1105 1110 1115 1120

Leu Lys Asn Leu Lys Lys Lys Lys Leu Met Thr Asp Arg Asn Ser Glu

1125 1130 1135

Glu Leu Cys Glu Leu Val Lys Val Met Phe Glu Tyr Lys Ala Leu Glu

1140 1145 1150

Claims

1. A method for rapidly detecting African swine fever virus based on CRISPR-Cas13a protein is characterized by comprising the following steps: the method comprises the following steps:

step (1) preparing a Cas13a protein;

designing and synthesizing primers suitable for RPA amplification;

designing and synthesizing crRNA suitable for the Cas13a protein in the step (3);

designing and synthesizing an RNA (ribonucleic acid) reporter molecule;

designing and synthesizing positive plasmids;

preparing positive plasmids;

step (7), preparing an amplification detection system;

and (8) detecting.

2. The method for rapidly detecting the African swine fever virus based on the CRISPR-Cas13a protein according to claim 1, wherein the method comprises the following steps: the step (1) specifically comprises the following steps:

3. The method for rapidly detecting the African swine fever virus based on the CRISPR-Cas13a protein according to claim 1, wherein the method comprises the following steps: the step (2) is specifically as follows: designing an upstream primer and a downstream primer for specifically detecting the African swine fever virus according to a conserved African swine fever virus VP72 gene, performing blast on the designed upstream primer and downstream primer in NCBI, and displaying the sequence of the African swine fever virus as a specific sequence;

the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1;

the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2;

4. The method for rapidly detecting the African swine fever virus based on the CRISPR-Cas13a protein according to claim 1, wherein the method comprises the following steps: the step (3) is specifically as follows: according to the characteristics of the Cas13a protein, crRNA for specifically recognizing African swine fever virus is directly designed in the sequence of the amplified gene fragment, blast is carried out in NCBI, and the sequence is shown to be a specific African swine fever virus sequence; the nucleic acid sequence of the crRNA is shown as SEQ ID NO. 3.

5. The method for rapidly detecting African swine fever virus based on CRISPR-Cas13a protein according to claim 4, wherein the method comprises the following steps: the step (4) is specifically as follows: designing an RNA reporter molecule according to the characteristics of Cas13a protein, wherein the sequence of the RNA reporter molecule is a fluorescent reporter group-TUUUUC-fluorescent quencher group, and the fluorescent reporter group is FAM, HEX, TET, JOE or VIC; the fluorescence quenching group is BHQ1, BHQ2 or BHQ 3.

6. The method for rapidly detecting the African swine fever virus based on the CRISPR-Cas13a protein according to claim 5, wherein the method comprises the following steps: the sequence of the RNA reporter molecule is FAM-TUUUUC-BHQ 1, the 5 'end is modified with FMA fluorescent group, and the 3' end is modified with BHQ1 quenching group.

7. The method for rapidly detecting the African swine fever virus based on the CRISPR-Cas13a protein according to claim 1, wherein the method comprises the following steps: the step (5) is specifically as follows: the nucleotide sequence of the African swine fever virus VP72 gene is cloned into pUc57 plasmid, the cloning site is SamI, the resistance is ampicillin resistance, and the cloning strain is DH5 alpha.

8. The method for rapidly detecting the African swine fever virus based on the CRISPR-Cas13a protein according to claim 1, wherein the method comprises the following steps: the step (6) is specifically as follows: inoculating the positive plasmid obtained in step (5) into LB medium containing 100. mu.g/ml ampicillin in an amount of 1%, and culturing overnight at 37 ℃ and 120rpm in a shaker; and (3) sucking bacterial liquid, extracting plasmids by adopting a Tiangen plasmid extraction kit, measuring the concentration by using Nanodrop 2000, calculating the copy number, and performing serial gradient dilution for subsequent use.

9. The method for rapidly detecting the African swine fever virus based on the CRISPR-Cas13a protein according to claim 1, wherein the method comprises the following steps: the amplification detection system in the step (7) comprises the following components and dosage:

10. the method for rapidly detecting the African swine fever virus based on the CRISPR-Cas13a protein according to claim 1, wherein the method comprises the following steps: the step (8) is specifically: and (3) preparing a recombinant plasmid working standard substance according to the positive plasmid prepared in the step (6), adding the working standard substance into the amplification detection system and the RPA reaction unit in the step (7), testing the reaction by a normal-temperature isothermal amplification fluorescence detector, and observing the result.