Establishment Of Detecting Method For Gosling Plague Virus

Gosling plague(Gosling, the Plague,GP) caused by the Parvoviridae, parvovirus goose parvovirus(the Goose Parvovirus, of GPV) is a gosling acute or subacute septic infectious disease. The disease mainly affects the goslings and Muscovy Duck in less than30days old. It has characteristics of rapid spread, incidence and death rate. The mortality rate is from90%to100%.And the morbidity and mortality rates declined as gosling days of age increased.1. This trial established a nested PCR diagnosis of gosling plague virus, which amplified the length of the expected size of515bp of the gene fragment. The specific test proved that only the gosling plague virus can be amplified specific bands. The sensitivity tests showed that when nucleic acid content of the gosling plague virus was0.03264fg/l, this PCR method was able to detect the virus and amplify a clear and specific fragment. The sensitivity of the PCR method was much higher than the conventional PCR for gosling plague virus. The false positive rate was also low.2. According to the Goose Parvovirus B strain gene sequence published in the Genbank the test designed specific primers of the VP3protein, the major structural protein, against the conserved region of VP3gene. Using the GPV DNA of Yangzhou separation the expected1605bp gene fragment has been amplified, and was purified to connect with pMD19-T vector,and then transformed into Escherichia coli cells DH5a. After enzyme cute and PCR, it was verify that the cloned gene was VP3gene of GPV. The PCR product was cloned into Pcold I and then transformed into DH5α. After positive bacteria identified the positive plasmid was transformed into BL21and induced by IPTG. VP3protein was purified by affinity chromatography and could react with the rehabilitation of gosling plague.3. By optimizing each step of the reaction condition, an indirect ELISA method for detecting VP3antibody was established, the purified recombinant VP3protein as an antigen. Ultimately the optimal antigen-coated concentration was0.0625μg/mL. The best serum dilution was1:40. The the critical value criteria for OD450nm was0.145. This ELISA method has good specificity, reproducibility and sensivity, and can be used for the epidemiological investigation of goose parvovirus,...