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Method for identifying wild strain and vaccine strain of hog cholera virus

A technology of swine fever virus and wild strains, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, which can solve the limitation of popularization and application of fluorescent quantitative PCR detection methods, and cannot achieve high-throughput detection, not suitable for rapid identification and other problems, to achieve the effect of shortening the detection time, fast detection speed and high accuracy

Active Publication Date: 2013-09-25
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the specificity of PCR detection technology is high, this method analyzes different subgroups according to the size of the electrophoretic bands of PCR amplification products, so this method is not yet capable of high-throughput detection, and the price of subgroup-specific probes Expensive also limits the popularization and application of fluorescent quantitative PCR detection method
Molecular sequencing analysis can also be used to distinguish vaccine strains from wild strains, but this method is time-consuming, labor-intensive, and expensive, and is not suitable for rapid identification

Method used

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  • Method for identifying wild strain and vaccine strain of hog cholera virus
  • Method for identifying wild strain and vaccine strain of hog cholera virus
  • Method for identifying wild strain and vaccine strain of hog cholera virus

Examples

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Embodiment 1

[0063] In order to verify the feasibility and reliability of the method of the present invention, a standard plasmid was constructed, each plasmid sequence was saved (correctly sequenced), and then the saved plasmid sequence was amplified for HRM analysis as a standard control.

[0064] (1) Construction of standard plasmid samples:

[0065] ① Take four wild strains identified as CSFV by sequencing (two strains are from a pig farm in Shanxi named D7, T144, and the other two strains are from a pig farm in Zhanjiang, Guangdong named Z1, Z2) and a vaccine strain ( The RNA of the cell seedlings produced by Guangdong Yongshun Biopharmaceutical Co., Ltd. (HCLV strain) was reverse transcribed into cDNA;

[0066] ②Amplify and purify the cDNA obtained by reverse transcription by PCR;

[0067] ③ Ligate the purified cDNA amplification products into the pMD-18T vector with a kit from TAKARA Company,

[0068] The carrier ligation reaction system (10 μl) is as follows:

[0069] ...

Embodiment 2

[0085] HRM detection of PCR products in clinical samples

[0086] (1) Extraction of CSFV RNA from disease samples:

[0087] Use the kit MiniBEST Viral RNA / DNA Extraction Kit Ver.4.0 to extract the CSFV RNA in the samples of the disease materials, the samples are blood, lymph nodes, liver, spleen, kidney and other tissues (the wild virus samples come from a pig farm in Shanxi and Guangdong , the vaccine strain was produced by Yongshun Biopharmaceuticals in Guangdong Province).

[0088] (2) One-step RT-PCR amplification of classical swine fever virus:

[0089] The extracted CSFV RNA was amplified by one-step RT-PCR using the PrimeScript one step RT-PCR Kit Ver.2 kit (including PrimeScript 1 step Enzyme Mix, 2×1 Step Buffer, etc.).

[0090] The reaction system is as follows: use the RNA extracted in (1) as a template, add 1-5 μl of template according to the actual concentration, add 0.5 μl of PrimeScript 1 step Enzyme Mix, add 0.5 μl of upstream and downstream primers P1 and P2...

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Abstract

The invention discloses a method for identifying a wild strain and a vaccine strain of a hog cholera virus. According to the method, a primer specially used for identifying the wild strain and the vaccine strain of the hog cholera virus is designed in a conservative region shared by the wild strain and the vaccine strain. The primer is high in specificity and can well multiply the wild strain and the vaccine low virulent strain; according to a reverse transcription-polymerase chain reaction technology and a high resolution melting (HRM) technology, the wild strain and the vaccine strain can be obviously identified. The method is a simple, quick and practical novel technology for identifying the wild strain and the vaccine strain of the hog cholera virus.

Description

technical field [0001] The invention relates to a method for distinguishing wild strains and vaccine strains of classical swine fever virus. Background technique [0002] Classical swine fever (CSF) is a contact and highly lethal disease caused by classical swine fever virus (CSFV), characterized by high fever retention, bleeding and high mortality in pigs. The World Organization for Animal Health lists it as a class A infectious disease. [0003] The CSFV genome is about 12.3kb long and is a single-strand linear positive-sense RNA consisting of 5'UTR, ORF and 3'UTR. ORF encodes a polyprotein consisting of 3898 amino acid residues. From the 5' end to the 3' end, the ORF of CSFV can translate 4 structural proteins (C, Ems, El, E2) and 8 non-structural proteins (Npro, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B ). Among them, the envelope glycoprotein E2 gene is the main antigen structural protein that induces the production of protective neutralizing antibodies; the 3'UTR of...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 郭鹏举张建峰张春红饶丹嘎利兵嘎沈海燕周恒朱余军郭翠丽
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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