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Avian Infectious Bronchitis(IB),caused by Avian Infectious Bronchitis Virus(IBV) of Coronavirus, and for chickens, charactered by cough, sneezing, tracheal rales; young chicken nasal fluid flow; The quantity and quality of laying decline, is an acute and highly contagious infectious disease which leads to significant economic loss in poultry industry. Vaccination is the main measures of prevention and control of avian infectious bronchitis, A variety of vaccine strains have different pathotypes or serotypes of the virus at home and abroad and vaccine efficacy is evaluated with a strong strain. Some strains are difficult to be identified with the traditional test method of seed viruses accurately, especially the same serotype strains, So it is necessary to establish the methods of molecular biology for further improving the IBV specific test.In this study, we used different era seed lots of H52stain、H120strain and M41strain,preserved by the research culture collection centers of China Institute of Veterinary Drug Control, as subjects, and obtain SI gene of32lots by RT-PCR using specific designed primers. After sequence analysis, the results show that: the1993.3.25and1993.3.28lots of H52strains were consisted with M41strain, through accessing to the original records, we found the viruses of two seed lots were failure. In safety inspection, the SPF chickens appeared rales, shakes and other sympotoms of virutent IBV. Compared to other several seed lots, Viruses of1983.10.7lot and1992.12lot have two and nine base virations respectively. So when propagating H52,do not use these two lots of seed viruses. There is not apparent bases variation in H120seed viruses. The seed viruses of M41strains can be divided into two groups: the gene sequences of1979.5.29、1988.4.8、1991.4.5、1993.4.6、1996.6.12lots are identified for M41strain; while the sequences of1976.9、1993.3.6、1993.3.22and2006.12lots are similar to H120strain.The study will then be download in GenBank H52strain,H120strain,M41strain, as well as popular abroad,4/91vaccine strain full genome sequence(total length of27.6kb) comparative analysis to find out the sequence differences. The use of self-designed four pairs of primers(primer A, primer B, primer C, primer D) of these four different genotypes of the strains were amplified. Primer A amplified the M41strain genome group sequence2898-4688bp at length for the1791bp the purpose of fragment while this pair of primers could not amplify the H52strain and H120strain, the minimum titer which required is103.5EID50/mL, the pair of specific primer can distinguish M41strain and H52strain, H120strain, B primers could amplify a fragment of1700bp in the whole genome sequence of21588bp-23288bp of the H52strain and H120strain, the minimum titer which required is103.5EID50/mL, then we use restriction endonuclease Nae I into PCR products, H120strain can be cut into two fragments of l000bp and700bp,while the H52strain amplification products does not exist the restriction sites, so can not be cut. Therefore, the use of specific primers B combined with restriction enzyme digestion method can differentiate between H52strain and H120strain. Using primers C can amplified fragment4/91strain S1gene sequences of length1351bp purpose fragment, the minimum titer which required is10.25EID50/mL, and combine with primer D can purify1066bp purpose fragment of4/91S1gene, but it can not amplify H52strain、n H120strain and M41strain, so we can effectively distinguish between4/91vaccine strain and H52strain,H120strain、M41strain. These three primers A、 B and C all have good specificity.Application of the established molecular biology identification method, this study we extracted a total of75batches IBV live vaccine of19vaccine companies from the National Vaccine Enterprise,15batches of IBV live vaccine from five foreign enterprises and buy back the11companies a total of16batches of IBV live vaccine from the market to do the pure analysis and identification. The result showed that H120strain vaccine and H52strain vaccine were detected using primer B, pure and are100%,did not find mistakenly mixed H52strain and H120strain, Vaccine using primer C and primer D inspection to detect nine batches of vaccine containing4/91, distribution in six companies from the75batches of vaccine domestic enterprises. From the market poultry vaccine we buy,we find1batch of vaccine containing4/91strain, and from foreign extraction live vaccines were not found4/91strain. To sum up, through the analysis of the IBV H52strain, H120strain,M41strain and popular foreign vaccine4/91strain, we establish avian infectious bronchitis virus molecular biology identification method, and further improve the seed virus specific test method. The method is then applied to the domestic and foreign markets in the avian infectious bronchitis virus live vaccine pure detection and provided basis for the future of IBV vaccine virus seed screening and vaccine efficacy assessment.
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