A triple PCR detection primer and kit for rapidly distinguishing African swine fever virus wild strains from gene-deleted strains

An African swine fever virus and gene deletion technology, applied in the field of virus strain identification, can solve the problems of cumbersome steps, unfavorable rapid identification and the like

Active Publication Date: 2021-05-04
SOUTH CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are cumbersome steps, which is not conducive to rapid identification

Method used

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  • A triple PCR detection primer and kit for rapidly distinguishing African swine fever virus wild strains from gene-deleted strains
  • A triple PCR detection primer and kit for rapidly distinguishing African swine fever virus wild strains from gene-deleted strains
  • A triple PCR detection primer and kit for rapidly distinguishing African swine fever virus wild strains from gene-deleted strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Primer Design

[0038]The inventor compared a large number of CD2V, P72, 360-505R genes of African swine fever virus (ASFV) published in the GenBanK database of NCBI (National Center for Biological Information), which are highly conserved and have specific regions, designed as ASFVCD2V, P72, 360 The -505R gene is a pair of specific primers, respectively named as P1, P2, P3, P4, P5, and P6, thereby providing a PCR primer for distinguishing ASFV wild-type strains and gene-deleted strains, specifically:

[0039] P1: ASFV-P72-F: 5'AACCAGTGGCCCTCTCCTAT 3' (SEQ ID NO: 1);

[0040] P2: ASFV-P72-R: 5'AATCGCATTGCCTCCGTAGT 3' (SEQ ID NO: 2);

[0041] P3: ASFV-CD2V-F: 5' TCCTAAGCCTTACAGTCGTTATCAGT 3' (SEQ ID NO: 3);

[0042] P4: ASFV-CD2V-R: 5'AGATAATGGCGGGATATTGGGTAGT 3' (SEQ ID NO: 4);

[0043] P5: ASFV-360-505R-F: 5' TCTTGTCCTTTTCATACGCCTCAT 3' (SEQ ID NO: 5);

[0044] P6: ASFV-360-505R-R: 5'GAGCACACCTGGGACCTCT 3' (SEQ ID NO: 6).

[0045] P1 and P2 are used to am...

Embodiment 2 3

[0048] Embodiment 2 Triple PCR detection method

[0049] Materials and Methods

[0050] 1.1 Primers in Example 1

[0051] 1.2 Sample DNA extraction

[0052] There are no special requirements for DNA extraction, which can be extracted according to conventional methods or DNA extraction kits. The extracted DNA was stored at -20°C for later use or immediately used for PCR amplification.

[0053] 1.3 Positive plasmid

[0054] Using the partial gene sequences of ASFV CD2V, P72, and 360-505R published in the GenBanK database to artificially synthesize the gene, connect it to the pJET1.2 cloning vector, transform Escherichia coli competent cell DH5α, and spread it on LB containing 100mg / L ampicillin On the culture medium plate, culture at 37°C for 12-16 hours. After the bacteria were picked, screened and identified by sequencing, the positive bacteria were expanded and cultured, and the plasmids were extracted. The positive plasmids were named pJET-P72, pJET-CD2V and pJET-360-505...

Embodiment 3

[0067] Embodiment 3 specificity test

[0068] According to the triple PCR detection method established in 1.4 in Example 2, pair 1: wild strain without gene deletion; 2: deletion of CD2V strain; 3: deletion of CD2V and 360-505R strain; 4: positive plasmid (P72); 5: negative Control (DEPC water); 6: classical swine fever virus (CSFV); 7: porcine pseudorabies virus (PRV); 8: porcine reproductive and respiratory syndrome virus (PRRSV); 9: porcine parvovirus (PPV); 10: Porcine encephalitis virus (JEV); 11: rotavirus (RV); 12: porcine epidemic diarrhea virus (PEDV); 13: porcine deltacoronavirus (PDCoV), for detection. The results are attached figure 2 shown.

[0069] From attached figure 2 As can be seen in the figure, corresponding to 1: wild strain without gene deletion; 2: deletion of CD2V strain; 3: deletion of CD2V and 360-505R strain; 4: positive plasmid (P72) in the corresponding fragment size. See clear bands, while negative control (DEPC water); 6: classical swine fe...

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Abstract

The invention discloses a triple PCR detection primer set for quickly distinguishing African swine fever virus wild strains from CD2V and / or 360-505R gene deletion strains. The nucleotide sequences of the three pairs of detection primers are as shown in SEQ ID NO: 1-6 shown. The present invention utilizes three pairs of primers to amplify three genes of African swine fever virus CD2V, P72, and 360-505R at one time, which reduces the detection cost and detection time of identifying different genes; and only needs one PCR reaction to amplify three different genes. The length of the gene, to identify whether there is a gene deletion in the strain. Only one PCR amplification is required to identify the three genes, and the cost is reduced by about 2 / 3 for the traditional method to separately amplify and detect the samples of the three genes, which has broad market prospects.

Description

technical field [0001] The invention relates to the technical field of virus strain identification methods, more specifically, to a triple PCR detection primer and a kit for quickly distinguishing African swine fever virus wild strains and gene deletion strains. Background technique [0002] African swine fever (ASF) is caused by African swine fever virus (ASFV), which can cause acute hemorrhagic fever, resulting in a large number of morbidity and mortality events (mortality close to 100%) in pigs. The World Organization for Animal Health (OIE) listed it as a must-report animal disease, and my country listed it as a first-class animal disease. Since the first case of African swine fever broke out in my country in 2018, African swine fever has caused major losses to my country's pig industry and seriously affected the healthy development of my country's pig industry. Studies at home and abroad have shown that African swine fever virus CD2V and 360-505R gene knockout can sign...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 沈永义陈瑞爱张旭
Owner SOUTH CHINA AGRI UNIV
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