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WO2020253537A1 - Procédé et kit de détection du virus de la peste porcine africaine - Google Patents

Procédé et kit de détection du virus de la peste porcine africaine Download PDF

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WO2020253537A1
WO2020253537A1 PCT/CN2020/094185 CN2020094185W WO2020253537A1 WO 2020253537 A1 WO2020253537 A1 WO 2020253537A1 CN 2020094185 W CN2020094185 W CN 2020094185W WO 2020253537 A1 WO2020253537 A1 WO 2020253537A1
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detection method
cas13a
rna
protein
crrna
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吴尧
杨海平
谢云鹤
李秋实
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Cure Genetics Co Ltd
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Cure Genetics Co Ltd
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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

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, which is caused by 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 detections are prone to occur.
  • the PCR method has a 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: 44314434;).
  • OIE routine laboratory diagnosis
  • 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.
  • the purpose of the present invention is to provide a rapid nucleic acid detection system and method for African swine fever virus based on the Cas13 protein.
  • a nucleic acid detection method for detecting African swine fever virus comprising the following steps:
  • RNA polymerase promoter at the 5'end
  • a detection system to the transcribed target sequence RNA of b for fluorescence detection.
  • the detection system includes one or more crRNA, Cas13 protein and single-stranded RNA nucleic acid probes, among which crRNA can correspond to the corresponding African swine fever virus gene Fragment combination.
  • RNA transcription system comprises RNA transcriptase, preferably T7 RNA transcriptase.
  • steps a, b, and c are performed in sequence; or three steps a, b, and c are performed simultaneously; or a and b are performed simultaneously, and then step c is performed in the system; or a After the steps, steps b and c are carried out simultaneously in the system.
  • steps a, b and c are performed in sequence; or after a is completed, steps b and c are performed simultaneously in the system.
  • step a RPA primers, RPA enzyme mixture containing recombinase and polymerase, MgOAc and buffer are added to the nucleic acid sample.
  • step a PCR primers, PCR enzyme mixture containing recombinase and polymerase and buffer are added to the nucleic acid sample.
  • a nucleic acid system for detecting African swine fever virus used in any one of the technical schemes 1-20, comprising the following components:
  • Fluorescence detection system contains (i) one or more crRNA, (ii) Cas13 protein, and (iii) single-stranded RNA nucleic acid probe, wherein crRNA can bind to the corresponding gene fragment of African swine fever virus.
  • RNA transcription system comprises RNA transcriptase, preferably T7 RNA transcriptase.
  • the Cas13 protein is selected from Cas13a, Cas13b or Cas13c, preferably Cas13a, and more preferably the Cas13a protein is derived from Leptotricia wadei; more preferably, the Cas13a protein has SEQ ID No : The sequence shown in 1, or the sequence with at least 80% identity with the sequence shown in SEQ ID No:1, or the sequence shown in SEQ ID No:1 with one or several amino acid substitutions, deletions and insertions A variant of the Cas13a protein.
  • amplification system a is an RPA amplification system, comprising an RPA primer, an RPA enzyme mixture containing a recombinase and a polymerase, MgOAc and a buffer.
  • amplification system a is a PCR amplification system, comprising PCR primers, a PCR enzyme mixture containing recombinase and polymerase, and a buffer.
  • nucleic acid sample is derived from pig tissue, which is preferably whole blood, pig plasma, pig serum, pig urine, pig saliva or pig oral mucosa .
  • the limitation on the preferred components in the technical solution 21 of the nucleic acid system for detecting African swine fever virus is the same as the limitation on the components in the technical solutions 1-20.
  • 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 targets and non-specific single-stranded RNA fluorescent probes after transcription of K205R gene after forming complexes between crRNA of different targets and Cas13a protein provided by the embodiments of the present invention
  • Figure 3 is a schematic diagram of the fluorescence detection results of Cas13a basic detection of different initial amounts of K205R gene provided by the embodiment of the present invention.
  • Figure 4 Sensitivity schematic diagram of the African swine fever virus detection method based on Cas13a and RPA provided by the embodiment of the present invention (different consecutive times);
  • Figure 5 Schematic diagram of the plasmid vector for LwaCas13a protein purification.
  • 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 the CRISPR/Cas effector protein.
  • Current research has found that there are two main types of CRISPR-Cas system.
  • the first type is Class 1 containing multi-subunit protein effector complexes and single-subunit protein effector complexes.
  • Class 2 where the Class 1 CRISPR-Cas system is the most common in bacteria and archaea (including all hyperthermophiles). This type of protein accounts for approximately 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 JA et 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 Cas13 (VI type CRISPR/Cas effector protein) protein family.
  • the Cas13 family includes Cas13a (C2c2), Cas13b (C2c4) and Cas13c (C2c7) (Koonin et al., Curr Opin Microbiol. 2017 June; 37: 67-78: "Diversity, classification and evolution of CRISPR-Cas systems").
  • Cas13a Cas13a
  • Cas13b Cas13b
  • Cas13c7 Cas13c protein involved in the technical scheme of the present invention
  • the Cas13 protein of the present invention is a Cas13a protein.
  • Cas13a protein (formerly known as "C2c2") refers to a crRNA-dependent endonuclease, which is a type VI enzyme in the CRISPR system classification.
  • the Cas13a protein of the present invention may be a Cas13a protein derived from different species, such as Leptotrichia shahii Cas13a, Lachnospiraceae bacterium MA2020 Cas13a, Lachnospiraceae bacterium NK4A179 Cas13a, Clostridium aminophilum (DSM 10710) Cas13a, Carnobacterium M.
  • DSM 10710 Cas13a, Carnobacterium M. Cas13a, Listeriaweihenstephanensis (FSLR9-0317) Cas13a, Listeriaceae bacterium (FSL M6-0635) Cas13a, Listeria newyorkensis (FSL M6-0635) Cas13a, Leptotricia wadei (F0279) Cas13a, Rhodobacter (Rhodobacter) capsulatus (SBcapsulatus (Rhodobacter 1003) Cas13a, Rhodobacter capsulatus (DE442) Cas13a, Leptotricia wadei (Lw2) Cas13a (LwaCas13a), or Listeria seeligeri Cas13a.
  • DSM 10710 Cas13a, Carnobacterium M. Cas13a, Listeriaweihenstephanensis (FSLR9-0317) Cas13a, Listeriaceae bacterium (FSL
  • the Cas13a protein is preferably LwaCas13a; more preferably it is Cas13a of the sequence shown in SEQ ID No:1, or has at least 80%, 83%, 85%, 86%, 87%, 88%, 89 with SEQ ID No:1. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical Cas13a, or with SEQ ID No:1 A variant of Cas13a with deletion, substitution or addition of several amino acids.
  • Cas13 proteins such as Cas13a, Cas13b, and Cas13c
  • 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 nucleoplasmin (for example, the nucleoplasmin dyad NLS with the sequence KRPAATKKAGQAKKKK); -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 the K205 gene sequence.
  • the crRNA of the present invention is a nucleic acid molecule that can combine with type VI CRISPR/Cas effector protein (Cas13 protein, such as Cas13a, Cas13b, Cas13c, etc.) to form a ribonucleoprotein complex (RNP) and target the complex to the target sequence RNA .
  • 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 VI 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 Cas13a to bind to the target RNA.
  • target RNA and target sequence RNA have the same meaning, and both refer to RNA gene fragments that need to be combined and detected by the CRISPR/Cas system of the present invention.
  • Cas13a protein After crRNA binds to Cas13 protein to form a ribonucleoprotein complex, when it performs its function, Cas13a protein recognizes the protospacer-adjacent motif (PAM) (non-G sequence) by single-base on the target sequence RNA When the appropriate PAM sequence is found, and the roughly complementary sequence to crRNA is identified, the target will be specifically cut.
  • PAM protospacer-adjacent motif
  • the guide sequence in the crRNA of the present invention is complementary to the target sequence of the target DNA.
  • the length of the guide sequence is 15-35 nucleotides (nt), for example, preferably 15-34nt, 15-32nt, 15-30nt, 15-28nt, 15-26nt, 15-24nt, 16-34nt, 16-32nt, 16-30nt, 16-28nt, 16-26nt, 16-24nt, 16-22nt, 17-34nt, 17-32nt, 17-30nt, 17-28nt, 17-26nt, 17- 24nt, 18-34nt, 18-32nt, 18-30nt or 18-28nt length).
  • the length of the guide sequence is 18-24 nucleotides (nt). In some cases, the guide sequence is at least 20 nt long (e.g., at least 20, 22, 24, or 26 nt 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.
  • 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.
  • Cas13 proteins such as Cas13a, Cas13b, Cas13c 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, 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 length).
  • the constant region length of crRNA is 30 or more.
  • 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 30 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 gene subtypes 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 2015Podlaskie, Warthog, Warmbaths, Tengani 62, Pretorisuskop 964, Mkuzi 1979, Malawi Lil-201 (1983), and isolate Kenya 1950.
  • the K205R gene (GeneID: 22220430) is one of the early transcribed 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 the target Dot the sequence to design crRNA.
  • the selected target sequence in the K205R gene is as follows:
  • Cas13a-crRNA target sequence ACTGCTGAAAGCAGATCTTGAAAAAACT (SEQ ID No: 9)
  • the target sequence 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, 196bp
  • cloned it into the pUC-57 plasmid (Table 2), using the plasmid extraction kit to extract the target gene plasmid DNA as a template, -80 Store at °C for later use.
  • the probe is a small piece of single-stranded RNA. Unlike ordinary 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 3-180 nt, and the preferred length is 5-30 nt.
  • the 5'end and 3'end of the single-stranded RNA probe are respectively labeled with a fluorescent group and a quenching group.
  • the fluorescent group can be, but not limited to, FAM (Carboxy fluorescein, green fluorescence), FITC (Fluorescein isothiocyanate), TET (Tetrachloro fluorescein, Tetrachloro fluorescein).
  • HEX Hydexachloro fluorescein
  • JOE 2,7-dimethyl-4,5-dichloro-6-carboxy fluorescein
  • rhodamine Rhodamine dyes such as R110 , TAMRA, Texas Red, etc.
  • ROX ROX
  • 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, ATTO 542, ATTO 550, ATTO565, ATTO Rho3
  • the quenching group may be, but not limited to, DABCYL, TAMRA, MGB, BHQ-0, BHQ-1, BHQ-2, BHQ-3 and the like.
  • the single-stranded RNA probe is 5-FAM/UUAUU/BHQ1.
  • the swine fever virus DNA is extracted; this extraction process can use conventional methods in the field or conventional extraction kits, For example, TaKaRa MiniBEST 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.
  • an RNA transcriptase promoter (e.g., T7 promoter) is added to the 5'end of a primer in a PCR or RPA reaction. This results in an amplified double-stranded DNA product that contains both the target DNA sequence and the RNA transcriptase promoter.
  • the amplified sample DNA (with RNA transcriptase promoter, such as T7 promoter) is used as a template, and a commercial RNA transcription kit is used to transcribe the amplified sample DNA into target sequence RNA.
  • RNA transcriptase promoter such as T7 promoter
  • the sample DNA is specifically amplified. You can first establish a PCR amplification system containing the sample DNA to be tested, including the sample DNA, PCR primers (one of which has an RNA polymerase promoter at the 5'end), PCR enzyme mixture, PCR buffer, etc.;
  • RNA transcription system commercial kit
  • NEB T7 transcription kit
  • RNA RNA sequence RNA to the detection system, which includes crRNA, Cas13a protein, single-stranded RNA nucleic acid probe, buffer, etc., and then perform fluorescence detection on it.
  • the detection system which includes crRNA, Cas13a protein, single-stranded RNA nucleic acid probe, buffer, etc.
  • PCR-based two-step method After the specific amplification of DNA in step 1 above is completed, the RNA transcription system in step 2 and the detection system in step 3 are added to the system at the same time.
  • the sample DNA is specifically amplified.
  • RNA RNA sequence RNA to the detection system, which includes crRNA, Cas13a protein, single-stranded RNA nucleic acid probe, buffer, etc., and then perform fluorescence detection on it.
  • the detection system which includes crRNA, Cas13a protein, single-stranded RNA nucleic acid probe, buffer, etc.
  • the sample DNA is specifically amplified.
  • the target sequence RNA is obtained after the reaction;
  • RNA RNA sequence RNA to the detection system, which includes crRNA, Cas13a protein, single-stranded RNA nucleic acid probe, buffer, etc., and then perform fluorescence detection on it.
  • the detection system which includes crRNA, Cas13a protein, single-stranded RNA nucleic acid probe, buffer, etc.
  • the sample DNA is specifically amplified.
  • RNA transcription commercial kit
  • detection system which includes T7 transcriptase, NTP buffer, crRNA, Cas13a protein, single-stranded RNA nucleic acid probe, detection buffer, etc., and directly Perform fluorescence detection on the system.
  • sample DNA After obtaining sample DNA from pig tissue or body fluid, directly establish a one-step amplification, transcription, and detection system, including sample DNA, RPA primers (one of which has an RNA polymerase promoter at the 5'end), and RPA enzyme mixture (among which Including recombinase and polymerase), MgOAc, RPA buffer, T7 transcriptase, NTP buffer, crRNA, Cas13a protein, single-stranded RNA nucleic acid probe, detection buffer, etc., and directly perform fluorescence detection on the system.
  • RPA primers one of which has an RNA polymerase promoter at the 5'end
  • RPA enzyme mixture among which Including recombinase and polymerase
  • 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 BioTek Cytation 3; in certain exemplary embodiments, the fluorescence detection system may be Thermo Varioskan TM LUX; in certain exemplary embodiments, the fluorescence detection system may be It is a fluorescent quantitative PCR. In some exemplary embodiments, the fluorescent detection system may be Applied Biosystems TM 7500 Real-Time PCR System.
  • pET21a-LwaCas13a Construction of expression vector: pET21a-LwaCas13a.
  • the following is a schematic diagram of the vector loop.
  • the LwaCas13a gene fragment (SEQ ID No. 37) is integrated into the pET21a vector (Jin Weizhi, see Figure 5) through XhoI and NdeI restriction endonucleases; the steps for expressing and purifying protein are as follows:
  • Steps The bacterial cells were collected by centrifugation at 8000 rpm for 30 minutes, resuspended by adding lysis solution, and crushed by a homogenizer. Centrifuge at high speed to separate the supernatant and precipitate. The supernatant passes through a 0.45 ⁇ m filter membrane and is ready for the next step of chromatographic purification.
  • Step: The nickel column (GE Life Sciences) is equilibrated with 0.5% solution B (20mm Tris, 250mM Nacl, 1M imidazole, 5% glycerol, pH 8.0).
  • AKTA instrument follows the instructions of the AKTA instrument to obtain a purified protein solution.
  • Step: Add the protein solution to the concentrated tube, centrifuge at 4°C, 5000rpm, 40 minutes, take out the concentrated tube, remove the penetrating solution, add 15ml protein storage solution (20mM Hepes, PH 7.5; 150mM KCl; 1% sucrose; 30%) Glycerol; 1mM dithiothreitol (DTT)), centrifugation at 4 degrees for 40 minutes; repeat the above steps 3 times to obtain the final LwaCas13a protein solution, which is divided into -80 degrees and stored.
  • 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.34) and downstream primer RPA-R( SEQ ID NO.35) sequence is shown in Table 3, one of the primers has a T7 transcription promoter sequence and a 6-base random sequence, and the primer is synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.;
  • 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.34) and downstream to the reaction system containing the lyophilized powder of RPA reaction enzyme primer RPA-R (SEQ ID NO.35) 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 280mM MgAc ion, add ddH 2 O to make up to 50ul, and react at 37°C for 15 min.
  • Example 2 after the Cas13a-crRNA and the Cas13a protein form a complex, the K205R gene is specifically cut and the ssRNA fluorescent probe is subjected to fluorescence detection of non-specific cut.
  • Cas13a and crRNA binding reaction in 10ul buffer (40mM Tris-HCl; 60mMNaCl; 6mM MgCl 2 ) add 10ul LwaCas13a protein at a concentration of 40uM and 20ul crRNA (Cas13a-crRNA) at a concentration of 20uM to prepare Cas13a- The final concentration of crRNA is 10uM system, incubate for 20min at room temperature;
  • Step 2 Add 5ul of Cas13a-crRNA complex in step 2) to each detection system, add 1ul 100nM of the amplified DNA (with T7 transcription promoter sequence) prepared in Example 1, 38ul of buffer (40mM Tris-HCl; 60mM NaCl; 6mM MgCl2), 1ul T7 transcriptase, 5ul MTP buffer (NEB), mix and add to RNaseAlert TM Substrate (Integrated DNA Technologies) containing single-stranded RNA fluorescent probe In, pipetting and mixing;
  • Cas13a-crRNA binding reaction Add 10ul LwaCas13a protein (40uM) and 20ul Cas13a-crRNA (20uM) in 10ul buffer (40mM Tris-HCl; 60mM NaCl; 6mM MgCl 2 ), and configure the final concentration of Cas13a and crRNA For a 10uM system, incubate for 20min at room temperature;
  • Step 2) Preparation of the detection system: Add 5ul of the Cas13a-crRNA complex in step 1) to the detection system, and add 1ul of the amplified DNA (with T7 transcription promoter sequence) prepared in Example 1 with different loads ( They are 10 7 , 10 8 , 10 9 , and 10 10 copies, respectively represented by E7, E8, E9, and E10), 1ul T7 transcriptase, 5ul MTP buffer (NEB), mixed and added to the fluorescent probe containing single-stranded RNA In the RNaseAlert TM Substrate (Integrated DNA Technologies) of the needle, pipette and mix;
  • Example 5 A method for detecting African swine fever virus based on the combination of Cas13a 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
  • Cas13a-crRNA binding reaction Add 10ul LwaCas13a protein (40uM) and 20ul Cas13a-crRNA (20uM) in 10ul buffer (40mM Tris-HCl; 60mM NaCl; 6mM MgCl 2 ), configure the final concentration of Cas13a and crRNA For a 10 ⁇ M system, incubate for 20min at room temperature;
  • Cas13a fluorescence detection reaction system Add 2.4ul each of upstream primer RPA-F (SEQ ID NO.34) and downstream primer RPA-R (SEQ ID NO.35) to 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), 3.5 ⁇ l 280mM MgAc ion, add single-stranded RNA fluorescent probe, add 5ul complex in step 1, 1ul T7 transcriptase, 5ul MTP buffer (NEB), 1ul RNase inhibitor (TaKaRa), add ddH 2 O to make up to 50ul;

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Abstract

La présente invention concerne un procédé de détection rapide du virus de la peste porcine africaine sur la base d'un complexe de protéines d'acides nucléiques CRISPR-Cas13a: sur la base de Cas13a et sous le guidage de l'ARNcr, l'ADN génique correspondant d'ASFV est spécifiquement clivé tandis qu'une caractéristique de l'activité de clivage d'ARNss non spécifique est produite pour la détection d'acides nucléiques.
PCT/CN2020/094185 2019-06-21 2020-06-03 Procédé et kit de détection du virus de la peste porcine africaine Ceased WO2020253537A1 (fr)

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