Primer group for simultaneously identifying wild strain and gene deletion strain of African swine fever based on multiple qPCR technology and test kit

An African swine fever and gene deletion technology, applied in recombinant DNA technology, microbial assay/test, biochemical equipment and methods, etc., can solve the problem of inability to distinguish between ASF wild virus strains and gene deletion vaccine strains, and achieve low cost , the effect of saving costs and reducing pollution

Inactive Publication Date: 2020-11-27
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the method of identifying ASFV mainly uses fluorescent quantitative PCR technology to detect the p72 gene, which cannot distinguish ASF wild strains from gene-deleted vaccine strains

Method used

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  • Primer group for simultaneously identifying wild strain and gene deletion strain of African swine fever based on multiple qPCR technology and test kit
  • Primer group for simultaneously identifying wild strain and gene deletion strain of African swine fever based on multiple qPCR technology and test kit
  • Primer group for simultaneously identifying wild strain and gene deletion strain of African swine fever based on multiple qPCR technology and test kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] 1. Design of specific primers and specific probes: Download multiple ASFV full genes (HLJ / 2018, SY18, N10 / 2018, AnhuiXCGQ) in Genbank, and use the BioEdit software package to perform Clustalw comparison. According to the p72 gene conservation of the above viruses Sequence, using Primer 5.0 software to design specific TaqMan probes and primers. According to the deletion sequence of the attenuated live vaccine strain HLJ / 18-7GD of the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (HLJ / 2018 strain deletion 73394-74476 nucleotides (CD2v) and 27942-35500 nucleotides (MGF) ) and the ASFV gene deletion vaccine strain (SY18 strain lacks 73368-74452 nucleotides (CD2v) and 27942-35500 nuclear (MGF)) consensus deletion fragment, using Primer 5.0 software to design specific TaqMan probes and primers for CD2v gene and MGF gene.

[0060] 2. Synthesis of primers and probes: synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0061] 3. See...

Embodiment 2

[0072] Multiplex real-time fluorescence method validation

[0073] 1. Specificity Verification

[0074] Utilize kit method of the present invention to respectively with the DNA of ASFV wild strain DNA and gene deletion vaccine strain DNA, porcine parvovirus (PPV), porcine pseudorabies virus (PRV) and porcine circovirus type 2 virus (PCV2) and pig breeding The specificity of primers and probes was verified by multiplex real-time fluorescent PCR amplification with the cDNA of respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) as templates. The results are shown in Table 3, and the results show that the designed primers and probes of the present invention have strong specificity.

[0075] Table 3 specificity verification test

[0076]

[0077] 2. Sensitivity evaluation

[0078] Quantify the positive standard products of ASFV p72 gene, CD2v gene and MGF gene to 107 copies / μL respectively, and serially dilute them by 10 times to 1.0×10 6 , 1.0×10 ...

Embodiment 3

[0081] Clinical suspicious sample detection

[0082] The multiple real-time fluorescent PCR detection method established by the present invention to simultaneously identify wild African swine fever strains and gene deletion vaccine strains detects 12 clinically suspicious samples, and the sample types include pig lungs, lymph nodes, tissues and serum. At the same time, positive samples were subjected to ordinary PCR detection and parallel sequencing analysis.

[0083] The results are shown in Table 4, and the results show that the method established by the present invention is consistent with the ordinary PCR detection results and sequencing results, and the method is accurate and reliable.

[0084] Table 4 Test results of clinical samples

[0085]

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Abstract

The invention provides a primer group for simultaneously identifying a wild strain and a gene deletion strain of African swine fever based on a multiple qPCR technology and a test kit, and belongs tothe technical field of virus detection. The primer probe group for simultaneously identifying the wild strain and the gene deletion strain of the African swine fever based on the multiple qPCR technology comprises a p72 gene specific primer probe, a CD2v gene specific primer probe and an MGF gene specific primer probe. The test kit comprises the primer probe group. Multiple qPCR detection is performed by adopting the test kit; p72 gene specific primers of a conserved gene of ASFV can amplify the wild strain and the gene deletion vaccine strain; specific primers of a CD2v gene and an MGF gene can only amplify the wild strain; and the three pairs of primers are combined for use, so that the wild strain and the gene deletion vaccine strain of the ASFV can be simultaneously identified. The primer group and the test kit have the advantages of accurate detection, sensitivity, high efficiency, low cost and the like, and have relatively high practical value for diagnosis of clinical samples and the breeding industry.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a primer set and a kit for simultaneously identifying African swine fever wild strains and gene deletion strains based on multiple qPCR technology. Background technique [0002] African swine fever (African Swine fever, East African Swine fever, ASF) is an acute, febrile and highly contagious viral disease of pigs, characterized by a short course of disease, but a mortality rate of up to 100%. The clinical manifestations of sick pigs were fever, cyanosis of the skin, and obvious bleeding of lymph nodes, kidneys, and gastrointestinal mucosa. African swine fever virus (ASFV) is an important member of the African swine fever family African swine fever virus genus. The diameter of the virus particle is 175-215nm, which is icosahedral and has a capsule. The genome is double-stranded linear DNA with a size of 170-190kb. At present, ASFV can be divided into 24 geno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/70C12Q1/686
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2561/113C12Q2563/107C12Q2545/114C12Q2537/143
Inventor 于江吴家强孟凯张玉玉刘娜孙文博陈智任素芳
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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