Establishment Of Double RT-PCR Detection Method Of CSFV And BVDV And Genetic Evolution Analysis Of E2 Gene

Classical swine fever(Classical Swine Fever Virus, CSFV), bovine viral diarrhea(Bovine Viral Diarrhoea Virus, BVDV) belong to the Flaviviridae Pestivirus genus members, which have similar structural and antigenically, generated cross immunological the reaction and the cross protection. Since both can infected porcines, seriously affected the healthy development of animal husbandry. The CSFV more infected porcines, the BVDV can infect not only livestock and small ruminants, including pregnant sows and piglets that most susceptible, the pathological changes and clinical symptoms similar with chronic classical swine fever. This is not only bring some troubles for disease prevention and control and difficult with judgment this disease From the view of nucleotide homology, the homology of nucleotide of BVDV and CSFV were about 60%, due to the presence of cross-reactivity in serological, which easily confused with swine fever and the diagnosis of disease bring influence. In recent years, clinical manifestations of classical swine fever was atypical, the emergence of the so-called "atypical swine fever", "mild swine fever" and "infected sow syndrome", the pathological examination of these so-called atypical swine fever and classical swine fever were not the same too. There are two main reasons, one hand may be long-term prevention and control of vaccine in our country, so that immune tolerance of classical swine fever virus in immune stress, alter some virulence and antigens, on the other hand may be infected CSFV and BVDV simultaneously, both exacerbated the harm of the virus, our cultivate was been catastrophic strike. So the status that porcine infected with BVDV must have a clear understanding and should take the necessary precautions, in vaccine production process, to avoid contamination of BVDV vaccine. According to this vary from the current classical swine fever, and the symptoms of two viruses were similar, which clinically difficult differential diagnosis by symptoms of two viruses. Timely differential diagnosis of two viruses contribute to farmers prevent and control the two viruses later, it is necessary to establish a methods to identify CSFV and BVDV simultaneously. Therefore, This article research the following aspects:(1)Use MGEA4.0 software comparison analysis of the nucleotide sequences of CSFV, BVDV, and seek their specific conserved region and design their own specific primers;(2)The optimization of the annealing temperature during the PCR process, optimization CSFV, BVDV optimum annealing temperature respectivey;(3)The use of optimize annealing temperature, it aim to be specific detection for CSFV, BVDV and several common viruses of farms;(4)By changing the concentration of CSVFand BVDV, detection capable of amplifying minimum concentration of nucleic acid of the CSFV, BVDV, the verification of the sensitivity of the primer;(5)By double RT-PCR testing, final verification the result of amplified with specific primers of CSFV, BVDV is consistent with the expected results.