Development And Application Of NS Gene PCR And A Monoclonal Antibody Based Competitive ELISA To Detect Goose Parvovirus Infection

Goose parvovirus infection also known as Gosling plague or Derzsy's disease, mainly infects 4-day-old to 20-day-old domestic goslings and Muscovy duckings. It can cause chordapsus and also liver, kidney, heart and other organs septic lesions, particularly the fibrous of the small intestine. The short course of the disease, rapid transmission, and a high mortality rate, causing serious economic losses, it's one of the important infectious diseases of goose cultural industry. The disease spread every year in different degree that hinders the healthy development of goose cultural industry, requires an accurate and convenient diagnostic method for diagnosis.A pair of primers was designed in conservative NS region according to all the sequences of seven goose parvovirus strains in Genbank. Nucleic acids of GPV-98E strain which was extracted from allantoic fluid of goose embryo serve as template and a PCR assay was developed based on optimization of reaction conditions. The sensitivity of the PCR reached 4.194pg for GPV nucleic acids and 0.15ELD50 for allantoic fluid of goose embryo respectively. Using the PCR assay, the specific fragment of 476bp was amplified from DNA sample of GPV only, which agreed with expectation, but not amplified from duck plague virus, avian influenza virus and goose paramyxovirus. All the specific fragments were amplified by the assay from 52 samples of goslings infected clinical with GPV including heart, liver and intestine, indicating the PCR assay developed for GPV was sensitive and specific,which had potential clinical value.The monoclonal antibodies (McAbs) against goose parvovirus were obtained by fusing myeloma cells SP2/0 and spleen cells after six weeks-old BALB/c mice were immunized with goose parvovirus 98E by ultracentrifugation three times in this study. Four hybridoma cell strains against goose parvovirus were obtained by screening with the indirect ELISA and 4-6 times subcloning, named 1G3,2G8,3B8 and 3B11, respectively. 1G3 and 3B11 were identified to be IgG2b ,and the others to be IgM. The results revealed that the first two hybridoma cell strains could secret steadily in vitro the McAbs against goose parvovirus, 3B8 secreted McAbs unsteadily. Western blot and IFA showed that the four McAbs could combine with the natural GPV. Blocking ELISA showed that 1G3 and 3B11 could be blocked by GPV positive serm.VP2-C-ELISA was established based on preliminarily pured rVP2-VLPs as antigen and 1G3 as competitive antibody, and theirs optimal reactive conditions were determined. The detection method could monitor antibody level after GPV-VLPs immuning and provided foundation and technical support for the rapid diagnosis, disease surveillance, epidemiological investigation and immune antibody titer determination.