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Double detection method of porcine reproductive and respiratory syndrome virus and bovine viral diarrhea virus from pig

A technology for bovine viral diarrhea and respiratory syndrome, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve problems such as poor sensitivity and long time

Inactive Publication Date: 2011-01-05
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0005] At present, conventional detection methods for PRRSV and BVDV infection have shortcomings such as long time and poor sensitivity, but double PCR can amplify multiple target genes in the same reaction system, which is fast, sensitive and efficient, and is a molecule of great significance. Biological detection method

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  • Double detection method of porcine reproductive and respiratory syndrome virus and bovine viral diarrhea virus from pig
  • Double detection method of porcine reproductive and respiratory syndrome virus and bovine viral diarrhea virus from pig
  • Double detection method of porcine reproductive and respiratory syndrome virus and bovine viral diarrhea virus from pig

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Embodiment Construction

[0038] 1 Primer design and synthesis

[0039] According to the published gene sequence of bovine viral diarrhea virus (GenBank accession number is AF220247.1), comparative analysis, the gene sequence of the 5' end non-coding region with strong conservation was selected, and two pairs of specificity were designed and synthesized by using the computer software Primer5. Primers B1 / B2, N1 / N2 (see Table 2-1).

[0040] Table 4-1 PCR primers for amplifying PRRSV ORF7 and BVDV5'UTR genes

[0041]

[0042] 2 RNA extraction and cDNA synthesis

[0043] 2.1 RNA extraction Take 150 μL of PRRSV virus-infected cell culture (Marc-145 purchased from ATCC) into EP tubes, add 600 μL of TRIZOL reagent, shake vigorously, let stand at room temperature for 10 minutes, add 200 μL of chloroform, shake vigorously, stand at room temperature for 5 minutes, Centrifuge at 12000rpm at 4°C for 15min, take the supernatant into a new EP tube, mix gently with 700μL of -20°C pre-cooled isopropanol, let stan...

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Abstract

The invention relates to a double RT-PCR detection method of porcine reproductive and respiratory syndrome virus (PRRSV) and bovine viral diarrhea virus from a pig (BVDV), belonging to the biotechnology field. A pair of gene specific primers are respectively designed aiming to the PRRSV and the BVDV, the double RT-PCR detection method of the BVDV and the PRRSV is established, and minimum amounts of cDNA detection of the PRRSV and the BVDV are respectively 3.8*10<-4>ng and 7*10<-4>ng. Samples of a lung, a spleen, a lymph gland and the like of 75 piglets of Jiangsu Province are detected through the method to have a result that 55 are PRRSV positive and 14 are BVDV positive, wherein 12 PRRSVs and 12 BVDVs are mixedly infected, the detection result coincidence rate of the PRRSV and a single RT-PCR and the detection result coincidence rate of the BVDV and the single RT-PCR are respectively 89.3 percent and 92 percent, thereby proving that the established double RT-PCR detection method can be used for detecting the BVDV and the PRRSV in a clinical sample. Compared with a single detection method, the double RT-PCR detection method can save the time and the cost of an experiment and enhance the detection efficiency.

Description

1. Technical field [0001] The invention relates to a double RT-PCR detection method for porcine reproductive and respiratory syndrome virus and porcine bovine viral diarrhea virus, which belongs to the high-tech field of biotechnology, and is specially used for the detection of PRRSV and BVDV infection in pigs, and can be commercialized Statistics of BVDV contamination in swine fever vaccine. 2. Background technology [0002] In recent years, PRRSV has become one of the most serious pathogens affecting the global swine industry [2] , the virus mainly causes reproductive disorders such as fever, anorexia, premature labor, late abortion, stillbirth, weak piglets and mummified fetuses in pregnant sows, as well as respiratory symptoms and high mortality in piglets. The disease is a viral disease that occurred in developed countries in the pig industry in the late 1980s, and has caused huge economic losses to the world's pig industry. [3] , has become one of the main diseases o...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 王克伟姜平李玉峰
Owner NANJING AGRICULTURAL UNIVERSITY
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