CN114391046A

CN114391046A - Method and kit for detecting African swine fever virus

Info

Publication number: CN114391046A
Application number: CN202080045110.3A
Authority: CN
Inventors: 吴尧; 杨海平; 谢云鹤; 李秋实
Original assignee: Suzhou Jingrui Biotechnology Co ltd
Current assignee: Suzhou Jingrui Biotechnology Co ltd
Priority date: 2019-06-21
Filing date: 2020-06-03
Publication date: 2022-04-22
Also published as: WO2020253537A1

Abstract

The invention provides a method for rapidly detecting African swine fever virus based on CRISPR-Cas13a nucleic acid protein complex, which comprises the following steps: nucleic acid detection is carried out based on the characteristic that Cas13a specifically cuts corresponding gene DNA of ASFV under the guide of crRNA and can generate nonspecific ssRNA cutting activity.

Description

Method and kit for detecting African swine fever virus

Technical Field

The invention belongs to the field of virus detection, and particularly relates to a system, a detection method and a kit for detecting African swine fever virus. The invention can be used for detecting African swine fever virus or other viruses capable of infecting pigs.

Background

African Swine Fever (ASF) is an acute highly infectious disease with clinical symptoms such as hemorrhagic fever caused by African Swine Fever Virus (ASFV), and the fatality rate is higher than 90%. ASFV is a linear double-stranded DNA virus, the genome length is about 170 kb-193 kb, and 150-200 proteins can be coded. ASFV is mainly transmitted by circulating infection among wild pigs, domestic pigs and soft ticks, and causes serious economic burden to the pig industry in disaster areas due to extremely high infectivity and lethality. ASF is listed by the world health Organization (OIE) as one of the legal reports of animal epidemics and is listed as a type of animal infectious disease in China. ASF was first discovered in Africa in the last 60 years and later spread to the continents of Europe, America, etc., more than 60 different strains of ASFV were found and currently classified into 24 genotypes according to the P72 gene. In 8 months in 2018, the Shenyang city in China is diagnosed with the first ASF epidemic situation, and about 400 infected pigs die in one month after the epidemic situation occurs. Subsequently, China's provinces and regions discover the ASF epidemic situation one after another, and the control of the ASF epidemic situation is reluctant.

Since no effective ASF vaccine and treatment method is found at present, the rapid and accurate diagnosis and screening method for early stage and even latent stage of epidemic disease discovery is very critical for the prevention and control of epidemic diseases. For the inspection and quarantine and rapid diagnosis of ASFV, there is no standard method and mature product on the market, mainly based on clinical phenotype and laboratory method for diagnosis and confirmation. The existing laboratory diagnosis method mainly focuses on three major directions of virus separation and identification, antigen-antibody immunity detection, virus nucleic acid PCR detection and the like. The virus separation and identification method belongs to the traditional gold standard diagnosis method, but the process is complicated, time-consuming and labor-consuming, and is usually used as a verification diagnosis method in a laboratory. The antigen-antibody-based immunodetection comprises direct or indirect fluorescent antibody detection and enzyme-linked immunosorbent assay detection, and although the process is simple, the sensitivity is not enough, and false negative and missed detection are easy to occur. The PCR method has certain sensitivity and specificity, is a popular detection method on the market, and comprises conventional PCR recommended by OIE for laboratory routine diagnosis and a fluorescent real-time quantitative PCR method (Ag ü ero M, et al.J. Clin Microbiol 2003, 41: 44314434;). However, there are reports of studies showing that mismatch of nucleotides between the primer and the viral target gene in the OIE recommended method leads to a decrease in PCR sensitivity and specificity (Gallado C et al.J. Clin Microbiol.2015 Aug; 53 (8): 2555-65). With the continuous update of PCR technology and the emergence of more research reports on ASFV, some new ASFV detection technology and method are also gradually emerging. Wang et al reported that about 100 copies of recombinant plasmid DNA molecules containing ASFV P72 gene segment could be detected within 10min by a rapid ASFV detection method based on Recombinase Polymerase Amplification (RPA) (Can J Vet Res.2017 Oct; 81 (4): 308-312). In 2018, Massimo Biagetti et al also reported a biosensor-based ASFV rapid detection method (Talanta.2018Jul 1; 184: 35-41). Although the sensitivity of results reported by the technologies is high, the technologies have a way to practical application and are not convenient and efficient, so that new convenient and efficient detection methods for rapid detection and early screening of ASFV are urgently needed in the market.

In 2012, scientists discovered for the first time a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene editing technique. The CRISPR-Cas system is an RNA-guided adaptive immune system within a microorganism, when a virus invades a bacterium, the bacterium is able to capture fragments of foreign genetic material and integrate them into the CRISPR sequence in its own genome. CRISPR RNA (crRNA) generated by transcription of the CRISPR sequence can be combined with CRISPR-binding protein (Cas nuclease), and provides binding and cleavage specificity for the Cas nuclease through base pairing with a target nucleic acid sequence (Cong et al science 2013.339: 819. sup. 823). Cas9 nuclease was the first Cas protein widely accepted and applied for genome editing, but scientists have now expanded the range of Cas proteins to more other types of Cas proteins, such as the newly reported Cas13a, Cas12a and Cas14a proteins, among others. Cas13a nuclease cleaves specific target RNA under the action of crRNA, and activates the activity of self nuclease to cleave nonspecific RNA.

Zhang Pioneer et al developed the SHERLLOCK system for nucleic acid detection based on Cas13a and RPA isothermal amplification technology, and performed rapid and accurate detection on Zika and Dengue viruses (Zhang et al science 2017 Apr 28; 356 (6336): 438-442). The Jannever et al research group found that Cas12a and other V-type CRISPR-Cas12 enzymes have the characteristic of target-activated non-specific ssDNase cleavage, and a DETECTR nucleic acid detection method is created by combining Cas12a with isothermal amplification, which realizes ultrahigh sensitivity of DNA detection and can rapidly and specifically detect human papillomavirus in a patient sample (Doudna JA et al science 2018 Apr 27; 360 (6387): 436-.

At present, the prevention and control of African swine fever viruses are greatly challenged, and in China and even all over the world, no product can be conveniently and efficiently used for rapid detection and early screening of ASFV, and particularly no diagnosis and early screening type product developed based on gene editing enzyme technology exists.

Disclosure of Invention

The invention aims to provide a nucleic acid rapid detection system and a nucleic acid rapid detection method of African swine fever virus based on Cas13 protein.

The invention provides the following technical scheme:

1. a nucleic acid detection method for detecting African swine fever virus comprises the following steps:

a. amplifying a nucleic acid sample, wherein one of primers used for amplification is provided with an RNA polymerase promoter at the 5' end;

b. adding an RNA transcription system into the step a, and transcribing the amplification product into target sequence RNA;

c. b, adding a detection system into the transcribed target sequence RNA of the b for fluorescent detection, wherein the detection system comprises one or more crRNA, Cas13 protein and a single-stranded RNA nucleic acid probe, and the crRNA can be combined with a corresponding gene fragment of the African swine fever virus.

2. The detection method according to claim 1, wherein the African swine fever virus genes are K205R, CP530R, CP204L and P72, preferably K205R.

3. The detection method according to

claim

1 or 2, wherein the Cas13 protein is selected from Cas13a, Cas13b or Cas13c, preferably Cas13a, more preferably Cas13a protein derived from Leptotrichia wadei.

4. The detection method of claim 3, wherein the Cas13a protein has the amino acid sequence of SEQ ID No: 1; or with SEQ ID No: 1 has at least 80% identity; or with SEQ ID No: 1 having one or several amino acid substitutions, deletions and insertions.

5. The detection method of claim 4, wherein the Cas13a protein is homologous to the protein of SEQ ID No: 1 has at least 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

6. The assay according to any one of the preceding claims, wherein the African swine fever virus gene fragment has a length of at least 15bp, preferably at least 20 bp.

7. The detection method according to any one of the preceding claims, wherein the guide sequence of the crRNA has a length of 15nt to 28 nt.

8. The assay of any one of the preceding claims, wherein the crRNA has a length of 29 to 100 nt.

9. The detection method according to claim 8, wherein the crRNA has a length of 40 to 50 nt.

10. The assay of any one of the preceding claims wherein multiple single stranded crrnas bind to a fragment of the same african swine fever virus gene, preferably multiple crrnas bind to a fragment of the K205 gene.

11. The method of any one of the preceding claims, wherein the plurality of single-stranded crrnas bind to a fragment of a gene of a different african swine fever virus, preferably the plurality of crrnas bind to a fragment of two or more of the K205R, CP530R, CP204L, and P72 genes.

12. The detection method according to any one of the preceding claims, wherein the 5 'end and the 3' end of the single-stranded DNA fluorescent probe are labeled with a fluorophore and a quencher, respectively.

13. The assay of any one of the preceding claims wherein the RNA transcription system comprises an RNA transcriptase, preferably T7RNA transcriptase.

14. The detection method according to any one of the preceding claims, wherein the amplification system is an RPA amplification system.

15. The detection method according to any one of claims 1 to 13, wherein the amplification system is a PCR amplification system.

16. The detection method according to claim 14, wherein the steps a, b and c are performed in order; or the three steps a, b and c are carried out simultaneously; or a and b are carried out simultaneously, after which step c is carried out in the system; or after the step a, simultaneously carrying out the steps b and c in the system.

17. The detection method according to claim 16, wherein the three steps a, b and c are performed simultaneously in the system.

18. The detection method of claim 15, wherein the steps a, b and c are performed sequentially; or after a is finished, simultaneously carrying out b and c steps in the system.

19. The detection method according to claim 14 or 16 or 17, wherein in step a, an RPA primer, an RPA enzyme mixture comprising a recombinase and a polymerase, MgOAc, and a buffer are added to the nucleic acid sample.

20. The detection method according to claim 15 or 18, wherein in step a, a PCR primer, a PCR enzyme mixture comprising a recombinase and a polymerase, and a buffer are added to the nucleic acid sample.

21. A nucleic acid system for detecting african swine fever virus according to any one of claims 1-20, comprising the following components:

a. a system for amplifying a nucleic acid sample, wherein one of the primers used for amplification carries an RNA polymerase promoter at its 5' end;

an RNA transcription system;

c. fluorescence detection system: comprising (i) one or more crRNAs, (ii) a Cas13 protein, and (iii) a single-stranded RNA nucleic acid probe, wherein the crRNAs are capable of binding to a corresponding gene fragment of African swine fever virus.

22. The system of claim 21, wherein said RNA transcription system comprises an RNA transcriptase, preferably a T7RNA transcriptase.

23. The system of claim 21 or 22, wherein the Cas13 protein is selected from Cas13a, Cas13b or Cas13c, preferably Cas13a, more preferably Cas13a protein is derived from Leptotrichia wadei; more preferably, the Cas13a protein has the amino acid sequence of SEQ ID No: 1, or a sequence corresponding to SEQ ID No: 1, or a sequence having at least 80% identity to the sequence set forth in SEQ ID No: 1 having one or several amino acid substitutions, deletions and insertions.

24. The system of any one of claims 21-23, wherein the amplification system a is an RPA amplification system comprising RPA primers, an RPA enzyme cocktail comprising a recombinase and a polymerase, MgOAc, and a buffer.

25. The system of claim 24 wherein said components b and c are in the same solution system.

26. The system of any of claims 21-23, wherein the amplification system a is a PCR amplification system comprising PCR primers, a PCR enzyme mixture comprising a recombinase and a polymerase, and a buffer.

27. The system of claim 26 wherein said components a, b, and c are in the same solution system; or components a and b are in the same solution system; or components b and c are in the same solution system.

28. The system of any of claims 21-27, wherein the nucleic acid sample is derived from a tissue of a pig, preferably whole blood, porcine plasma, porcine serum, porcine urine, porcine saliva, or porcine oral mucosa.

The preferable limitations of the components in the above nucleic acid system for detecting African swine fever virus according to claim 21 are the same as the limitations of the components in claims 1 to 20.

The method for rapidly detecting the African swine fever virus based on the gene editing enzyme comprises a basic detection system and a detection system after amplification, and compared with the prior art, the method has the following advantages:

high sensitivity: the application of the invention can realize aM-level DNA detection with the detection limit as low as 10 copies, can realize the early screening of ASFV besides the rapid qualitative detection of ASFV, and has extremely low omission factor caused by false negative;

high specificity: when the detection time of the microplate reader is as short as 15min, the change of fluorescence value is analyzed, and meanwhile, the comparison result of multiple crRNA targets is combined, so that the false positive is reduced to the maximum extent, and the specificity is enhanced;

-fast: the invention can complete detection within half an hour;

-convenience: the one-step detection of a plurality of reagents in a single tube is realized, the operation is convenient, and the steps are simple;

-versatility: the invention can realize the rapid detection of DNA viruses such as ASFV and the like.

Drawings

FIG. 1: the result of 2% agarose gel electrophoresis of the product of the RPA reaction using the synthetic K205R gene plasmid DNA as the template provided by the embodiment of the invention is shown schematically (with T7 and without T7);

FIG. 2: the embodiment of the invention provides a schematic diagram of fluorescence detection results after cutting a specific target and a non-specific single-stranded RNA fluorescent probe after transcription of a K205R gene after a complex is formed by crRNA of different targets and Cas13a protein;

FIG. 3: the embodiment of the invention provides a schematic diagram of the fluorescence detection result of Cas13a basic detection on K205R genes with different initial amounts;

FIG. 4: the sensitivity schematic diagram (continuous different time) of the African swine fever virus detection method based on Cas13a and RPA provided by the embodiment of the invention;

FIG. 5: schematic representation of LwaCas13a protein purification plasmid vector.

Detailed description of the invention

The invention is described in detail herein by reference to the following definitions and examples. The contents of all patents and publications, including all sequences disclosed in these patents and publications, referred to herein are expressly incorporated by reference.

CRISPR/Cas system

CRISPR refers to clustered, regularly interspaced short palindromic repeats (clustered regular intercarried palindromic repeats) that are the immune system of many prokaryotes.

Cas proteins refer to CRISPR/Cas effector proteins, and it has been found that there are two main classes of CRISPR-Cas systems, the first Class being Class 1 comprising multi-subunit protein effector complexes and Class 2 comprising single-subunit protein effector complexes, wherein the Class 1CRISPR-Cas system is most common in bacteria and archaea (including all hyperthermophiles), which accounts for about 90% of all the identified CRISPR-Cas proteins (Makarova, et al. an updated evaluation of CRISPR-Cas systems. Nat. Rev. Microbiol. 2015; 13: 722-736). The Class 2 CRISPR-Cas system is almost exclusively present in bacteria, and Cas proteins available for this system mainly include type II, V, VI effector proteins, which account for about 10% of CRISPR-Cas proteins, mainly the commonly used Cas9 protein (type II), and newly discovered Cas12 (type V), Cas13 (type VI), Cas14 (type V) proteins, etc. (Chylinski K et al nucleic Acids Res.2014; 42: 6091. sup. 6105; Shmakov S, et al. mol. cell. 2015; 60: 385. 397; Sergey Shmakov et al Nat Rev Microbiol.2017 March; 15 (3): 169. sup. 182; Doudna JA. science.2018. Nov 16; 362 (6416): 839. sup. 842). The relatively simple effector complex formation of the Class 2 CRISPR-Cas system makes it one of the hottest gene editing tools for scientific research.

In the present application "CRISPR/Cas system" and "CRISPR system" are the same meaning.

Cas protein

The Cas protein related to the technical scheme of the invention is a Cas13(VI type CRISPR/Cas effector protein) protein family. The Cas13 family includes Cas13a (C2C2), Cas13b (C2C4), and Cas13C (C2C7), among others (Koonin et al, Curr Opin Microbiol.2017 June; 37: 67-78: "Diversity, classification and evaluation of CRISPR-Cas systems"). These Cas proteins may originate from different genera, and their enzymatic activities may also differ significantly.

In a specific embodiment, the Cas13 protein of the invention is a Cas13a protein. The Cas13a protein (old called "C2C 2") refers to a crRNA-dependent endonuclease, which is a type VI (type VI) enzyme in CRISPR system classification. The Cas13a protein of the invention may be a Cas13a protein of different species origin, such as Leptospiria shahii Cas13a, Lachnospiraceae bacteria MA2020 Cas13a, Lachnospiraceae bacteria NK4A179 Cas13a, Clostridium amiophilum (DSM 10710) Cas13a, Carnobacterium gallinarum (DSM 4847) Cas13a, Paslebacterium proponicigenes (WB4) Cas13a, Listeria newyolkengenscensis (FSLR9-0317) Cas13a, Listeriobacter (FSL M6-0635) Cas13a, Listeria newyolkenstis (FSL M6-0635) 13a, Leptospiria (F379) Cas a protein, Rhodiola 5913, Cas 5913, Rhodiola 5913 Cas 5913 (Cas 5913) or Rhodiolabacter sphaericalis (FSLR 5913) Cas 5913, Rhodiola 202) Cas 5913, Rhodiola 5913, Cas 5913 of Rhodiola 5913, Cas 2 or Rhodiola 5913. The Cas13a protein is preferably LwaCas13 a; more preferably SEQ ID No: 1, or Cas13a corresponding to the sequence shown in SEQ ID No: 1, Cas13a having at least 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, or is a variant of SEQ ID No: 1a variant of Cas13a with a deletion, substitution or addition of one or several amino acids.

In some cases, Cas13 proteins, e.g., Cas13a, Cas13b, Cas13c, are fusion proteins. In some cases, the heterologous polypeptide (fusion partner) provides subcellular localization, i.e., the heterologous polypeptide contains subcellular localization sequences (e.g., Nuclear Localization Signal (NLS) for targeting the nucleus, Nuclear Export Sequence (NES) for maintaining the sequence of the fusion protein outside the nucleus, sequences that maintain the fusion protein retained in the cytoplasm, mitochondrial localization signal for targeting mitochondria, chloroplast localization signal for targeting chloroplasts, golgi localization signal, etc.). In some cases, the type V CRISPR/Cas effector protein (e.g., Cas13 protein) does not include an NLS, and thus the protein does not target the nucleus. Non-limiting examples of NLS include NLS sequences derived from: an NLS of SV40 virus large T antigen having the amino acid sequence PKKKRKV; NLS from nucleoplasmin (e.g., nucleoplasmin dyad NLS with sequence KRPAATKKAGQAKKKK); c-myc NLS has the amino acid sequence PAAKRVKLD or RQRRNELKRSP; the hRNPA 1M 9 NLS has the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY. In some cases, the heterologous polypeptide can provide a tag (i.e., 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 xhis tag; Hemagglutinin (HA) tag; FLAG tag; Myc tag; biotin tag; ceravidin tag; etc.). The fusion protein, in some cases, may comprise one or more targeting sequences or tags, each tag or targeting sequence may be one or more repeats.

Identity of each other

The correlation between two amino acid sequences or nucleotide sequences is described by the parameter "identity".

For The purposes of The present invention, The degree of identity between two amino acid sequences or nucleotide sequences is determined using The Needle program, such as The EMBOSS Software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al 2000, Trends in Genetics 16: 276-. Optional parameters used are gap penalty of 10, gap extension penalty of 0.5 and EBLOSUM62 substitution matrix (EMBOSS version of BLOSUM 62). The output of the Needle labeled "longest identity" (obtained using the-nobrief option) is used as the percent identity and is calculated as follows:

(same residue X100)/(length of alignment-total number of gaps in alignment).

crRNA

The crRNA of the present invention is CRISPR RNA, and is a single-stranded guide RNA that directs the Cas protein to specifically bind to the K205 gene sequence. The crRNA of the invention is a nucleic acid molecule that binds to type VI CRISPR/Cas effector proteins (Cas13 proteins, e.g., Cas13a, Cas13b, Cas13c, etc.) to form a ribonucleoprotein complex (RNP) and targets the complex to a target sequence RNA. In some embodiments, the crRNA may be a hybrid DNA/RNA such that the crRNA includes DNA bases in addition to RNA bases. The crRNA of the present invention includes a guide sequence (also referred to as a "spacer," which is hybridizable to the target sequence DNA), and a constant region (a region adjacent to the guide sequence and binding to the type VI CRISPR/Cas effector protein). The "constant region" may also be referred to herein as a "protein binding segment". In some embodiments, e.g., for Cas13a, the constant region is at the 5' end of the crRNA. In the present invention, crRNA is a nucleic acid that can guide Cas13a to bind to a target RNA. The terms "target RNA" and "target sequence RNA" are used synonymously in this application and refer to the RNA gene fragments that need to be bound and detected by the CRISPR/Cas system of the present invention.

After the crRNA binds to the Cas13 protein to form a ribonucleoprotein complex, the Cas13a protein recognizes a protospacer-adjacent motif (PAM) (non-G sequence) during the line-play function, and by single-base recognition of the target sequence RNA, specific cleavage of the target is not performed until a sequence that is approximately complementary to the crRNA is recognized after the appropriate PAM sequence is found.

The guide sequence in the crRNA of the present invention is complementary to the target sequence of the target DNA. In a specific embodiment, the length of the guide sequence is 15 to 35 nucleotides (nt), for example, preferably 15 to 34nt, 15 to 32nt, 15 to 30nt, 15 to 28nt, 15 to 26nt, 15 to 24nt, 16 to 34nt, 16 to 32nt, 16 to 30nt, 16 to 28nt, 16 to 26nt, 16 to 24nt, 16 to 22nt, 17 to 34nt, 17 to 32nt, 17 to 30nt, 17 to 28nt, 17 to 26nt, 17 to 24nt, 18 to 34nt, 18 to 32nt, 18 to 30nt, or 18 to 28nt in length. In particular embodiments, the guide sequence is 18-24 nucleotides (nt) in length. In some cases, the guide sequence is at least 20nt long (e.g., at least 20, 22, 24, or 26nt long). In some cases, the guide sequence is at least 23 nucleotides. In some cases, the guide sequence is at least 25 nucleotides. In some cases, the guide sequence is at least 28 nucleotides.

In some cases, the guide sequence has 80% or more (e.g., 85% or more, 90% or more, 95% or more, or 100%) complementarity to the target sequence of the target DNA. In some cases, the guide sequence is 100% complementary to the target sequence of the target DNA. In some cases, the target DNA comprises at least 15 nucleotides (nt) complementary to the guide sequence of the guide RNA.

Examples of constant regions of guide RNAs that can be used with type VI CRISPR/Cas effector proteins (Cas13 proteins, e.g., Cas13a, Cas13b, Cas13c) are shown in table 1.

Table 1: constant region of crRNA

In specific embodiments, the crRNA comprises a nucleotide sequence having a constant region with 70% or greater identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% identity) to the constant region of any one of the crRNA sequences set forth in table 1. In certain embodiments, the constant region of the crRNA comprises or has a nucleotide sequence set forth in table 1.

In some embodiments, the constant region comprises a complementary RNA sequence that forms an RNA duplex (dsRNA) by self-folding.

In some cases, the constant region of the crRNA is 15 or more nucleotides (nt) in length (e.g., 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, 35 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 or more, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, or 48 or more nt in length). In some cases, the constant region of the crRNA is 30 or more in length. In some cases, the constant region of the crRNA ranges in length from 12 to 100nt (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 constant region of the crRNA ranges from 28 to 100nt in length. In some cases, the length of the constant region at the 5' end of the crRNA ranges from 28 to 40 nt.

In some cases, 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 30 or more nucleotides (nt) in length (e.g., 34 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, or 80 or more in length). In some cases, the crRNA is 35 or more in length

African swine fever virus

African Swine Fever Virus (ASFV) is a single-molecule linear double-stranded DNA virus, belongs to the African swine fever virus family, and is the only member of the family at present. ASFV viruses have been found to have 24 gene subtypes (Galindo & Alonso, 2017; Quembo et al, 2018), and the genomic sequences have been determined by genome sequencing. The method of the invention can detect all the discovered strains related to 24 ASFV gene subtypes.

The 24 known subtypes of ASFV virus include BA71V, Ken05TK1, strain E75, Georgia 2007/1, Ken06, strain L60, Benin 971, 26544OG10, NHV, OURT 883, 47Ss2008, R35, N10, R25, R7, R8, ASFV 2015Podlaskie, Warthog, Warmbaths, Tengani 62, Pretoriuskop 964, Mkuzi 1979, Malawi Lil-201(1983), and isolate Kenya 1950.

K205R gene

The K205R gene (GeneID: 22220430) is one of early transcription genes in ASFV genome, the total length is 618bp, the pK205R protein coded by the gene can stimulate the body to produce antibody early; the invention designs crRNA by taking a relatively conserved fragment in the K205R gene as a target sequence.

In a specific embodiment, the sequence of the target site in the K205R gene is selected as follows:

cas13a-crRNA target sequence ACTGCTGAAAGCAGATCTTGAAAAAACT (SEQ ID No: 9)

In a specific embodiment, the target sequence has a length of at least 15 bp. The K205R gene fragment refers to a sequence of at least 15bp, preferably at least 20bp, in length in the K205R gene. Artificially synthesizing and selecting a fragment sequence (namely, a target sequence, 196bp) of the African swine fever virus K205R gene, cloning the fragment sequence into a pUC-57 plasmid (shown in a table 2), extracting a target gene plasmid DNA by using a plasmid extraction kit as a template, and storing at-80 ℃ for later use.

TABLE 2

Single-stranded RNA fluorescent probe

The probe is a short single-stranded RNA fragment, and unlike conventional probes, the sequence of the RNA fragment can be any sequence, and does not need to be complementary to the target RNA. The length of the single-stranded RNA probe can be 3-180nt, and the preferred length is 5-30 nt.

The 5 'end and the 3' end of the single-stranded RNA probe are respectively marked with a fluorescent group and a quenching group. The fluorescent group may be, but is not limited to, FAM (carboxyfluorescein, green fluorescence), FITC (Fluorescein isothiocyanate), TET (Tetrachloro-6-carboxyfluorescein, Tetrachloro fluoroscein), HEX (Hexachloro-6-methylfluorescein, Hexachloro fluoroscein), JOE (2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein), rhodamines (Rhodamine dyes such as R110, TAMRA, Texas Red, etc.), ROX, Alexafluor dyes (such as Alexa Red, etc.), and the like

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, ATTO 542, ATTO 550, ATTO565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho 5, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740), Dylight dyes, cyanine dyes (such as Cy2, Cy3, Cy3.5, Cy 3.3 b, Cy5, Cy5.5, Cy7, Cy 7.483 14), Cyo5, Cyo5.5, CyoFlugene dyes, Sequat dyes, and Squat dyes. The quenching group can be, but is not limited to DABCYL, TAMRA, MGB, BHQ-0, BHQ-1, BHQ-2, BHQ-3 and the like. For example, the single-stranded RNA probe is 5-FAM/UUAAUU/BHQ 1.

Sample DNA acquisition and amplification

Obtaining tissue of pig, such as whole blood, pig plasma, pig serum, pig urine, pig saliva, and pig oral mucosa, and extracting swine fever virus DNA; the extraction procedure may be carried out using methods conventional in the art or using conventional extraction kits, such as the TaKaRa MiniBEST virus RNA/DNA extraction kit, to extract CSFV DNA. The extracted sample DNA can be directly used for the detection of the invention, or under the condition that the sample DNA is small in quantity, the sample DNA is amplified and then the detection of the invention is carried out.

The nucleic acid amplification system of the present invention can be divided into two types: traditional PCR methods and isothermal amplification RPA methods.

In certain exemplary embodiments, conventional Polymerase Chain Reaction (PCR) can be used to amplify the sample DNA.

In certain exemplary embodiments, a Recombinase Polymerase Amplification (RPA) reaction can be used to amplify the sample DNA. The RPA reaction uses a recombinase capable of pairing a sequence-specific primer with a homologous sequence in a double-stranded DNA sample. If target DNA paired with the primer is present, DNA amplification is initiated without the need for additional sample manipulation, such as thermal cycling. The entire RPA amplification system is a stable dry formulation that can be safely transported without refrigeration. The RPA reaction may be carried out at isothermal temperature, with an optimal reaction temperature of 37-42 ℃. In order to amplify the sequence comprising the sample DNA to be detected, sequence specific primers can be designed.

In certain embodiments, an RNA transcriptase promoter (e.g., T7 promoter) is added 5' to one primer of a PCR or RPA reaction. This results in an amplified double stranded DNA product comprising both the target DNA sequence and the RNA transcriptase promoter.

RNA transcription

The amplified sample DNA (with an RNA transcriptase promoter, such as the T7 promoter) is used as a template and transcribed into the target sequence RNA using a commercial RNA transcription kit.

Nucleic acid detection method for African swine fever virus

Three-step PCR-based method:

1. after obtaining sample DNA from the tissue or body fluid of the pig, the sample DNA is specifically amplified. Firstly, a PCR amplification system containing sample DNA to be detected can be established, and the PCR amplification system comprises the sample DNA, PCR primers (one of which the 5' end is provided with an RNA polymerase promoter), a PCR enzyme mixture, a PCR buffer solution and the like;

2. adding the PCR amplification product to an RNA transcription system (commercial kit), and transcribing the amplification product into target sequence RNA using T7 transcription kit (NEB) according to the instructions;

3. and adding the transcribed target sequence RNA into a detection system, wherein the detection system comprises crRNA, Cas13a protein, a single-stranded RNA nucleic acid probe, a buffer solution and the like, and then carrying out fluorescence detection on the CRRNA.

Two-step PCR-based process: after the DNA specific amplification of the step 1 is completed, the RNA transcription system of the step 2 and the detection system of the step 3 are added to the system at the same time.

Three-step method based on RPA:

1. after obtaining sample DNA from the tissue or body fluid of the pig, the sample DNA is specifically amplified. Firstly, an RPA amplification system containing sample DNA to be detected can be established, and the system comprises the sample DNA, an RPA primer (one of which the 5' end is provided with an RNA polymerase promoter), an RPA enzyme mixture (which comprises recombinase and polymerase), MgOAc, RPA buffer solution and the like;

2. adding the RPA amplification product to an RNA transcription system (commercial kit), and transcribing the amplification product into target sequence RNA using T7 transcription kit (NEB) according to the instructions;

Two-step RPA-based process:

1. after obtaining sample DNA from the tissue or body fluid of the pig, the sample DNA is specifically amplified. Firstly, an RPA amplification and T7 transcription system containing sample DNA to be detected is established, the system comprises the sample DNA, an RPA primer (one of which the 5' end is provided with an RNA polymerase promoter), an RPA enzyme mixture (which comprises recombinase and polymerase), MgOAc, RPA buffer solution, T7 transcriptase, NTP buffer solution and the like, and target sequence RNA is obtained after reaction;

2. and adding the transcribed target sequence RNA into a detection system, wherein the detection system comprises crRNA, Cas13a protein, a single-stranded RNA nucleic acid probe, a buffer solution and the like, and then carrying out fluorescence detection on the CRRNA.

Or

2. the RPA amplification product is added into an RNA transcription (commercial kit) and detection system, which comprises T7 transcriptase, NTP buffer, crRNA, Cas13a protein, single-stranded RNA nucleic acid probe, detection buffer, etc., and the fluorescence detection of the system is directly performed.

One-step detection based on RPA:

after sample DNA is obtained from tissues or body fluid of a pig, a one-step method system for amplification, transcription and detection is directly established, wherein the one-step method system comprises the sample DNA, an RPA primer (one 5' end of the RPA primer is provided with an RNA polymerase promoter), an RPA enzyme mixture (comprising recombinase and polymerase), MgOAc, an RPA buffer solution, T7 transcriptase, NTP buffer solution, crRNA, Cas13a protein, a single-stranded RNA nucleic acid probe, detection buffer solution and the like, and the fluorescence detection of the system is directly carried out.

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 requires the following modules: 1) the temperature control module is used for controlling the temperature within the range of 0-100 ℃, preferably within the range of 25-50 ℃ and preferably at a constant temperature of 37 ℃; 2) the fluorescence detection module has excitation light wavelength of 490nm and emission light wavelength of 520 nm; or the wavelength of the exciting light is 535nm, and the wavelength of the emitting light is 560 nm; 3) the timing detection function can detect the set fluorescence every 0.5-120 minutes, preferably every 2-15 minutes, more preferably 2-5 minutes, and the duration is 10 minutes-3 hours, more preferably 15 minutes-2 hours.

In certain exemplary embodiments, the fluorescence detection system can be a BioTek rotation 3; in certain exemplary embodiments, the fluorescence detection system can beThermo Varioskan ^TMLUX; in certain exemplary embodiments, the fluorescence detection system may be fluorescent quantitative PCR, and in certain exemplary embodiments, the fluorescence detection system may be Applied Biosystems^TM 7500Real-Time PCR System。

Examples

The examples are given by way of illustration only and are not intended to limit the invention in any way.

The abbreviations have the following meanings: "h" refers to hours, "min" refers to minutes, "s" refers to seconds, "d" refers to days, "μ L" refers to microliters, "ml" refers to milliliters, "L" refers to liters, "bp" refers to base pairs, "mM" refers to millimoles, and "μ M" refers to micromoles.

Example 1: LwaCas13a protein purification

1 protein expression

1.1 expression vector construction: pET21a-LwaCas13 a. The following is a vector circular schematic diagram, the LwaCas13a gene fragment (SEQ ID No.37) was integrated into pET21a vector by XhoI and NdeI restriction endonucleases (King of King, see FIG. 5); the procedure for expressing the purified protein was as follows:

1.2 expression strain selection: BL21(DE3) (NEB)

1.3 culture conditions: shaking culture

Instrument consumables: shaking table, LB medium (Bio-Industrial, A507002)

The method comprises the following steps:

taking out the plate (BL21(DE3)) from a 4-degree refrigerator for activation, and standing at 37 degrees overnight; transferring: 500ml of LB inoculated with bacteria liquid 4ml, 37 degrees constant temperature culture for 4 hours, OD600 ═ 0.8, take out and put at room temperature, the temperature of the shaking table is reduced to 18 degrees, add 500ul of IPTG (isopropyl-beta-D-thiopyranoside, Tiangen RT108-01), overnight induction, induction time 20 hours.

2 protein purification

2.1 cell harvesting and lysis

Instrument consumables: centrifuge, homogenizer, high speed centrifuge, 0.45 μm filter (Mercury Miller, SLHV033), lysis buffer

The method comprises the following steps: the thalli is centrifugally collected at 8000rpm for 30 minutes, added with lysate for resuspension, and crushed by a homogenizer. The supernatant and the precipitate were separated by high-speed centrifugation. The supernatant was passed through a 0.45 μm filter and ready for further chromatographic purification.

2.2 chromatographic column purification process:

the instrument comprises the following steps: AKTA-purify, column, chromatography solution

2.2.1 Nickel column chromatography

The method comprises the following steps: the nickel column (GE life sciences) was equilibrated with 0.5% solution B (20mM Tris, 250mM Nacl, 1M imidazole, 5% glycerol, pH 8.0). Purified protein solutions were obtained following the AKTA instrumentation procedure.

2.2.2 heparin chromatography

The method comprises the following steps: this was equilibrated with 25% solution B (20mM Hepes, 1M Nacl, 5% glycerol, pH 7.5) using a heparin (Heprin) chromatography column (GE life sciences). Purified protein solutions were obtained following the AKTA instrumentation procedure.

3 concentrating and changing liquid

Instrument consumables: 30kD concentrator tube (Merck Millipop, UFC903096), low-temperature high-speed centrifuge

The method comprises the following steps: adding the protein solution into a concentration tube, centrifuging at 4 degrees for 40 minutes at 5000rpm, taking out the concentration tube, removing the penetration liquid, adding 15ml of protein preservation solution (20mM Hepes, pH 7.5; 150mM KCl; 1% sucrose; 30% glycerol; 1mM Dithiothreitol (DTT)), and centrifuging at 4 degrees for 40 minutes; and repeating the steps for 3 times to obtain a final LwaCas13a protein solution, subpackaging and storing at-80 ℃.

Example 2: RPA reaction with plasmid DNA of synthetic K205R gene as template

1) Selection of conserved region of K205R gene and gene cloning: performing homology comparison on the whole genome sequences of 24 ASFV virus strains (Table1) submitted and authenticated on GenBank, finding out a conserved region of K205R gene as an RPA amplified sequence (196bp), and analyzing the sites of variable bases of the 24 ASFVs on the gene segment (Table 2); the K205R gene fragment (SEQ ID NO.10) was directly synthesized and cloned into a pUC-57 vector (GenScriptCat. No. SD1176) to synthesize a plasmid pUC-57-K205R.

2) Design and synthesis of RPA primers: according to the design requirement of the RPA primer, the RPA primer comprises at most two variable base sites, the sequences of the designed upstream primer RPA-F (SEQ ID NO.34) and the designed downstream primer RPA-R (SEQ ID NO.35) are shown in Table 3, wherein one primer has a T7 transcription promoter sequence and a random sequence of 6 bases, and the primers are synthesized by Jinzhi biological science and technology Limited, Suzhou;

3) RPA reaction and agarose gel electrophoresis: diluting the above designed and synthesized primers to 10uM final concentration, adding 2.4ul of each of the upstream primer RPA-F (SEQ ID NO.34) and the downstream primer RPA-R (SEQ ID NO.35) into a reaction system containing RPA reaction enzyme lyophilized powder, 29.5ul of RPA buffer (provided in RPA kit), and 10ul of RPA buffer⁴(denoted by 1E 4) and 10⁸(indicated by 1E 8) different copy amounts of pUC-57-K205R plasmid DNA, 3.5. mu.l 280mM MgAc ion, plus ddH₂The O is fully supplemented to 50ul, and the reaction is carried out for 15min at 37 ℃. The reaction product of 5ul was aspirated and detected by electrophoresis in 2% agarose gel, and the results of gel imaging are shown in FIG. 1, which shows that the pair of primers can effectively perform RPA amplification on K205R gene plasmid DNA, and the length of the amplified product is 209 bp.

Example 3: validation of Cas13a-crRNA against K205R Gene

In this example 2, after Cas13a-crRNA forms a complex with Cas13a protein, fluorescence detection of specific cleavage of K205R gene and non-specific cleavage of ssRNA fluorescent probe was performed.

1) Preparation of crRNA: cas13a-crRNA was synthesized by Cincisco Biotech, Suzhou, with the RNA sequence (SEQ ID NO.36) shown in Table 3, and the synthesized RNA was diluted to 20uM and stored at-20 ℃ for further use.

2) Cas13a reacts with crRNA binding: in 10ul of buffer (40mM Tris-HCl; 60mM NaCl; 6mM MgCl)₂) Adding 10ul LwaCas13a protein with the concentration of 40uM and 20ul crRNA (Cas13a-crRNA) with the concentration of 20uM to prepare a system with the final concentration of both Cas13a-crRNA of 10uM, and incubating for 20min at room temperature;

3) preparation of a detection system: 5ul of Cas13a-crRNA complex from step 2) was added to each assay, 1ul of 100nM of the amplified DNA from example 1 (with the T7 transcription promoter sequence), 38ul of buffer (4)0mM Tris-HCl; 60mM NaCl; 6mM MgCl2), 1ul T7 transcriptase, 5ul MTP buffer (NEB), mixing well, adding into RNase alert containing single-stranded RNA fluorescent probe^TMIn substrate (integrated DNA technologies), blow-beating and mixing uniformly;

4) detection of fluorescent signal: and (3) quickly transferring the reaction solution to a 96-hole half-hole microplate reader plate for signal detection in the microplate reader: carrying out continuous kinetic fluorescence signal detection under the condition of Ex/Em-535/560 nM wavelength, collecting fluorescence signals once every 2min, and continuously monitoring for 1-2 h;

5) and (3) analyzing a fluorescence detection result: the results are shown in FIG. 2.

The results in fig. 2 show that the signal for fluorescence values of the Cas13a-crRNA set can show significant enhancement around 15 minutes for 100nM concentration of DNA, indicating that efficient cleavage is achieved.

Example 4 different initial amounts of K205R Gene for the Primary detection of Cas13a

1) Cas13a-crRNA binding reaction: in 10ul of buffer (40mM Tris-HCl; 60mM NaCl; 6mM MgCl)₂) Adding 10ul Lwa Cas13a protein (40uM) and 20ul Cas13a-crRNA (20uM) to configure a system with 10uM of final concentration of both Cas13a and crRNA, and incubating for 20min at room temperature;

2) preparation of a detection system: 5ul of Cas13a-crRNA complex from step 1) was added to the test lines, and 1ul of the amplified DNA prepared in example 1 (with T7 transcription promoter sequences) (10 each) was added at different loadings⁷、10 ⁸、10 ⁹、10 ¹⁰Copies, designated E7, E8, E9, E10), 1ul of T7 transcriptase, 5ul of MTP buffer (NEB), mixed well and added to RNase alert containing a single-stranded RNA fluorescent probe^TMIn substrate (integrated DNA technologies), blow-beating and mixing uniformly;

3) detection of fluorescent signal: and (3) quickly transferring the reaction solution to a 96-hole half-hole microplate reader plate for signal detection in the microplate reader: carrying out continuous kinetic fluorescence signal detection under the condition of Ex/Em-535/560 nM wavelength, collecting fluorescence signals once every 2min, and continuously monitoring for 1-2 h;

4) the result of the fluorescence detection is divided intoAnd (3) analysis: the results are shown in FIG. 3 for 10¹⁰Copy DNA, the signal of fluorescence value can show significant difference in 15 minutes or so; for 10⁹Copy DNA, the signal of fluorescence value can show significant difference at about 40 minutes; for lower loading of DNA, the signal of fluorescence value was not significantly different from NC group (negative control group without target DNA added);

TABLE 3

Example 5 African swine fever virus detection method based on combination of Cas13a and RPA, plasmid DNA of partial conserved region sequence of synthetic African swine fever virus K205R gene is used as target for detection

1) Cas13a-crRNA binding reaction: in 10ul of buffer (40mM Tris-HCl; 60mM NaCl; 6mM MgCl)₂) Adding 10ul Lwa Cas13a protein (40uM) and 20ul Cas13a-crRNA (20uM) to configure a system with the final concentration of both Cas13a and crRNA being 10uM, and incubating for 20min at room temperature;

2) cas13a fluorescence detection reaction system: adding 2.4ul each of upstream primer RPA-F (SEQ ID NO.34) and downstream primer RPA-R (SEQ ID NO.35) into a reaction system containing RPA reaction enzyme lyophilized powder, adding 29.5ul of RPA buffer (provided in RPA kit and purchased from TwistDx), adding 1ul of pUC-57-K205R plasmid DNA (10, 10) with different loads diluted by 10 times concentration gradient²、10 ⁴、10 ⁸Copy per microliter: indicated by E1, E2, E4, E8), 3.5. mu.l of 280mM MgAc ion, addition of single-stranded RNA fluorescent probe, addition of 5ul of the complex of step 1, 1ul of T7 transcriptase, 5ul of MTP buffer (NEB), 1ul of RNase inhibitor (TaKaRa), and addition of ddH₂Supplementing O to 50 ul;

3) and (3) quickly transferring the reaction solution to a 96-hole half-hole microplate reader plate for signal detection in the microplate reader: carrying out continuous kinetic fluorescence signal detection under the condition of Ex/Em-490/520 nM wavelength, collecting fluorescence signals once every 2min, and continuously monitoring for 1-2 h;

4) and (3) analyzing a detection sensitivity result: the results are shown in fig. 4, with fluorescence values increasing with increasing concentration of target and reaction time; the method can detect the plasmid DNA of 10 copies/ul African swine fever virus pUC-57-K205R, and the fluorescence value is obviously different from that of a negative control group without a template when the detection time is 30 min; for 10000 copies/ul African swine fever virus K205R plasmid DNA, the fluorescence value can show obvious difference after detection reaction for 15 min; the method has simple and quick process, the detection experiment period of 10 copies is about 1h long, and the detection experiment period of high loading capacity is as low as about 30-45 min.

Claims

A nucleic acid detection method for detecting African swine fever virus comprises the following steps:

a. amplifying a nucleic acid sample, wherein one of primers used for amplification is provided with an RNA polymerase promoter at the 5' end;

b. adding an RNA transcription system into the step a, and transcribing the amplification product into target sequence RNA;

c. b, adding a detection system into the transcribed target sequence RNA of the b for fluorescent detection, wherein the detection system comprises one or more crRNA, Cas13 protein and a single-stranded RNA nucleic acid probe, and the crRNA can be combined with a corresponding gene fragment of the African swine fever virus.
The test method according to claim 1, wherein the African swine fever virus genes are K205R, CP530R, CP204L and P72, preferably K205R.
The assay of claim 1 or 2, wherein the Cas13 protein is selected from Cas13a, Cas13b or Cas13c, preferably Cas13a, more preferably Cas13a protein is derived from Leptotrichia wadei.
The assay of claim 3, wherein the Cas13a protein has the amino acid sequence of SEQ ID No: 1;

or with SEQ ID No: 1 has at least 80% identity; or with SEQ ID No: 1 having one or several amino acid substitutions, deletions and insertions.
The assay of claim 4, wherein the Cas13a protein hybridizes to SEQ ID No: 1 has at least 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
The assay according to any one of the preceding claims, wherein the African swine fever virus gene fragment has a length of at least 15bp, preferably at least 20 bp.
The assay of any preceding claim, wherein the guide sequence of the crRNA has a length of 15nt to 28 nt.
The assay of any preceding claim wherein the crRNA has a length of 29-100 nt.
The assay method of claim 8, wherein the crRNA has a length of 40-50 nt.
The assay of any preceding claim, wherein multiple single-stranded crrnas bind to a fragment of the same african swine fever virus gene.
The assay of any preceding claim, wherein a plurality of single stranded crrnas bind to fragments of different african swine fever virus genes.
The detection method according to any one of the preceding claims, wherein the single-stranded DNA fluorescent probe is labeled with a fluorophore and a quencher at its 5 'end and 3' end, respectively.
The assay of any preceding claim wherein the RNA transcription system comprises an RNA transcriptase, preferably T7RNA transcriptase.
The assay of any preceding claim wherein the amplification system is an RPA amplification system.
The detection method according to any one of claims 1 to 13, wherein the amplification system is a PCR amplification system.
The assay of claim 14, wherein steps a, b and c are performed sequentially; or the three steps a, b and c are carried out simultaneously; or a and b are carried out simultaneously, after which step c is carried out in the system; or after the step a, simultaneously carrying out the steps b and c in the system.
The assay of claim 16 wherein steps a, b and c are performed simultaneously in the system.
The assay of claim 15, wherein steps a, b and c are performed sequentially; or after a is finished, simultaneously carrying out b and c steps in the system.
The assay of claim 14 or 16 or 17 wherein in step a, an RPA primer, an RPA enzyme cocktail comprising a recombinase and a polymerase, MgOAc and a buffer are added to the nucleic acid sample.
The detection method according to claim 15 or 18, wherein in the step a, a PCR primer, a PCR enzyme mixture comprising a recombinase and a polymerase, and a buffer are added to the nucleic acid sample.
A nucleic acid system for detecting African swine fever virus comprises the following components:

a. a system for amplifying a nucleic acid sample, wherein one of the primers used for amplification carries an RNA polymerase promoter at its 5' end;

an RNA transcription system;

c. fluorescence detection system: comprising (i) one or more crRNAs, (ii) a Cas13 protein, and (iii) a single-stranded RNA nucleic acid probe, wherein the crRNAs are capable of binding to a corresponding gene fragment of African swine fever virus.
The system of claim 21, wherein the RNA transcription system comprises an RNA transcriptase, preferably a T7RNA transcriptase.
The system of claim 21 or 22, wherein the Cas13 protein is selected from Cas13a, Cas13b or Cas13c, preferably Cas13a, more preferably Cas13a protein is derived from Leptotrichia wadei; more preferably, the Cas13a protein has the amino acid sequence of SEQ ID No: 1, or a sequence corresponding to SEQ ID No: 1, or a sequence having at least 80% identity to the sequence set forth in SEQ ID No: 1 having one or several amino acid substitutions, deletions and insertions.
The system of any one of claims 21-23, wherein the amplification system a is an RPA amplification system comprising RPA primers, an RPA enzyme cocktail comprising a recombinase and a polymerase, MgOAc, and a buffer.
The system of claim 24 wherein components b and c are in the same solution system.
The system of any one of claims 21-23, wherein the amplification system a is a PCR amplification system comprising PCR primers, a PCR enzyme mixture comprising a recombinase and a polymerase, and a buffer.
The system of claim 26, wherein said components a, b, and c are in the same solution system; or components a and b are in the same solution system; or components b and c are in the same solution system.
The system of any one of claims 21-27, wherein the nucleic acid sample is derived from a tissue of a pig, preferably whole blood, pig plasma, pig serum, pig urine, pig saliva, or pig oral mucosa; it can also be derived from the objects and environmental samples that have been exposed to swine.