Cloning Of Yanbian Strain VP3 Gene And Establishment Of A Single-tube Nested PCR Diagnosis Method For Gosling Plague Virus

Gosling Plague is caused by the gosling plague virus, which caused 4-20-day goslings and the young ducks an acute or subacute, a high degree contact, septic infectious diseases, it caused a serious harm for cultivation. The disease can be diagnosised by clinical symptoms and epidemiological characteristics of a preliminary, but it must be make use of etiology inspection, serological methods and molecular biology techniques to confirm final diagnosis. Etiology inspection and serological methods are lack of not enough sensitive and quick. The PCR technique with high sensitivity and specificity can meet the needs that diagnosis the clinical disease accurately and rapidly.A pair of specificity primers were designed for amplificated VP3 gene according to the republished gene sequence B of all genes in genbank for the research in its conserve zone, amplificated VP3 gene with Yanbian Strain genome DNA of the gosling plague virus. amplificated complete VP3 gene fragment in accordance with expectancy, after recovering and purifying, to insert pGEM-T Easy vector, then convert into intestine bacillus competent cell JM109 to clone, by enzyme incid analyses, PCR identification and sequence analysis, confirmed that was goose plague virus VP3 gene. At the same time, the established gosling plague single-tube nested PCR, amplificated 406bp length gene fragment in accordance with expectancy, specific test indicated that only gosling plague-virus-templates can amplifiy specific bands. Sens showed that it can still be detected when nucleic acid content of goose plague virus is 0.02175fg/μL, finding limpid specific fragment, it is sensitive as much 100 times as conventional PCR diagnostic method. 80 sick-dead goslings were tested by gosling plage single-tube nested PCR and conventional PCR diagnostic method, 20 goslings with typical pathological changes positive detection rate was 100% ( 20/20), 60 goslings without typical pathological changes positive detection rate was 93.3%(56/60), and 80%(48/60) by gosling plague single-tube nested PCR, and conventional PCR diagnostic method, it can be seen that single-tube nested PCR is more sensitive and specifical than conventional PCR diagnostic method, avoided the results of false negative by conventional PCR diagnostic method, providing an effective diagnostic mean for clinic accurately and rapidly.