The Establishment Of Detection Method For Porcine Parvovirus

The PPV 7909 and PPV NADL-2 were grown on PK-15 cell lines, harvested and concentrated against polyethylene glycol 6000 overnight after freezing, thawing and ultrasonication as 80% Cytopathic effect (CPE) appeared. This antigen was purified through the method of gel filtration with Sephadex G-200 . The hyperimmune serum of rabbit and guinea-pig anti-PPV was prepared by immunization in rabbit and guinea-pig with purified PPV. Mainly immunoglobulin G (IgG) of sheep anti-rabbit, rabbit and guinea-pig anti-PPV was obtained by ammonium sulphate precipitation subsequently unsalted with Sephadex G-25 and chromatography over diethylamino-ethyl-cellulose-32 ( DEAE-32 ) . The sheep anti-rabbit IgG conjugated to horseradish peroxidase and labeled with collodial gold was individually made by the method of sodium periodate and sodium citrate-tannic acid.Three immunology methods that indirect double antibody sandwish enzyme-linked immunosorbent assay (ELISA), Dot-ELISA and immunogold-silver staining (IGSS) were established by the optimal density fixation of rabbit, guinea-pig anti-PPV IgG and sheep anti-rabbit IgG according to the phalanx determination. Three methods proved to be superior in terms of specificity and duplication through the specificity test blocking test, cross test and duplication test. The Dot-ELISA could detect 0.1 HA- PPV, and indirect double antibody sandwish ELISA, IGSS could detect 0.05HA PPV as indicated by sensitivity test and compared with haemagglutination assay (HAT ).A pair of 20-base primers within PPV structure vp2 coding region was synthesized. The expected 158bp fragment was individually amplified through the decrease of time and cost of extracting DNA from PPV-infected PK-15 cells by the method of extraction with phenol/chloroform , twice extraction with phenol/chloroform and boiling. The amplified fragment was shown to be specific for PPV DNA as indicated by EcoR I digestion. The developed PCR could detect 0.008HA PPV nearly equal to 1 PFU trough the sensitivity tests.Twenty-six clinical specimens were detected by the methods of HAT, indirect double antibody sandwish ELISA, Dot-ELISA, IGSS and PCR. Ten out of 26 samples examined for the presence of PPV by PCR were positive and 16 were negative. Six out of 10 samples were positive by indirect double antibody sandwish ELISA and IGSS they had crown rump length less than 16cm, five were positive by Dot-ELISA and three were positive by HAT.