Fluorescent PCR (polymerase chain reaction) detection method for identifying traditional strain and highly pathogenic strain of PRRSV (porcine reproductive and respiratory syndrome virus)

A technology for highly pathogenic strains and respiratory disorders, applied in the field of biological detection, can solve the problems of inability to quantitatively measure samples, and achieve the effect of accurate identification

Inactive Publication Date: 2012-09-05
天津市动物疫病预防控制中心
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Problems solved by technology

The traditional PCR method cannot perform accurate quantitative determ

Method used

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  • Fluorescent PCR (polymerase chain reaction) detection method for identifying traditional strain and highly pathogenic strain of PRRSV (porcine reproductive and respiratory syndrome virus)
  • Fluorescent PCR (polymerase chain reaction) detection method for identifying traditional strain and highly pathogenic strain of PRRSV (porcine reproductive and respiratory syndrome virus)
  • Fluorescent PCR (polymerase chain reaction) detection method for identifying traditional strain and highly pathogenic strain of PRRSV (porcine reproductive and respiratory syndrome virus)

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Embodiment Construction

[0050] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but not as a limitation of the present invention.

[0051] 1. Preparation materials:

[0052] 1.1 Plasmids and strains

[0053] The plasmids (TJ-H1 nsp2-T and TJ-H3 nsp2-T) of the nsp2 genes of the highly pathogenic PRRSV strain TJ-H1 and the traditional strain TJ-H3 have been cloned by our laboratory (published in "Animal Medicine Progress", No. 31, 2010, paper titled: "Clone and genetic evolution analysis of PRRSV Tianjin isolate Nsp2 gene", and the two plasmids can be provided to the public), Genbank accession numbers are GQ923891, GQ923892, GQ923893 ( GQ923891 and GQ923892 are highly pathogenic strains TJ-H1, and the homology reaches 100%; GQ923893 is the traditional strain TJ-H3). Twelve sera positive for porcine reproductive and respiratory syndrome virus antibodies were provided by the Tianjin Animal Disease Prevention and Control Center...

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Abstract

The invention discloses a fluorescent PCR (polymerase chain reaction) detection method for identifying the traditional strain and highly pathogenic strain of PRRSV (porcine reproductive and respiratory syndrome virus). In order to accurately and quickly detect the nsp2 gene-deleted PRRSV, an upstream primer and two downstream primers are designed on the nucleotide sequence of the nsp2 gene, wherein one of the downstream primers is designed in a base sequence-deleted area. A PCR method for identifying the traditional strain and the highly pathogenic nsp2 gene-deleted strain is established through condition optimization; and a fluorescent quantitative PCR method for identifying the nsp2-deleted variant strain according to a melting curve is created by applying the optimal condition to the SYBR Green fluorescent quantitative PCR analysis, wherein the sensitivity is enough to detect 3-6 gene copies. Compared with the conventional PCR, the fluorescent quantitative PCR method disclosed by the invention can quickly and accurately detect the traditional strain and the deleted strain, and the sensitivity is 10 times higher than that of the conventional PCR.

Description

technical field [0001] The invention belongs to the technical field of biological detection and discloses a fluorescent PCR detection method for distinguishing traditional strains and highly pathogenic strains of porcine reproductive and respiratory syndrome virus. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as "PRRS", is a disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), characterized by reproductive disorders in sows and respiratory symptoms in piglets. symptoms, and a highly contagious disease characterized by high mortality. The disease broke out in the United States for the first time in 1987, and it has also become epidemic infection in various countries in the world, causing huge losses to the world's pig industry. The disease began to become popular in most provinces of my country in 2006. The so-called "swine unknown high fever" has caused huge losses to my country's pig industry. ...

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 李秀梅
Owner 天津市动物疫病预防控制中心
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