| PURPOSE: (1) to compare PCV2 antigen diagnostic methods. (2) to develop an indirect enzyme-linked immunosorbent assay(ELISA) for detecting PCV2 antibody. MEHTOUD: (1) 14 PCV2 PCR positive samples were treated regularly ,then inoculated onto PK-15 cells for three generations. They were identified by PCR. PCV2 strains were continued to propagate in the PK-15 cells from third generation to fifth generation with treatment of D(+)-glucosamine, and the influence of D(+)-glucosamine on the isolation and propagation of PCV2 was measured. (2) There were four methods for PCV2 purification .method1: differential velocity centrifugation ; method2: sucrose discontiguous grads centrifugation; method3: chloride cesium isodensity centrifugation; method4: chloroform extraction then ultracentrifugation. The best purification method of PCV2 was confirmed by comparing the effect of coating antigens which were puried by different methods. The best purified virus was used for coating antigen. The optimal conditions were determined for the PCV2-ELISA , such as the coating concentration, secondary antibody (HRP-SPA) dilution, serum sample dilution, blocking solution, the reactive time of HRP-SPA, the threshold, specificity reaction, repeatability test,.RESULTS: (1) Six PCV2 strains were isolated ,so the virus isolation rate was 42.83% (6/14) (2)six PCV2 strains (third generation) were continued to propagate in the PK-15 cell to fifth generation with treatment of D(+)-glucosamine. Four strains were harvested in treatment group, only one strains was harvested in the control. (3) contrast to the effect of coating antigens puried by four different methods, it's show that method4 was the best method for PCV2 purification. (4) The optimal conditions of PCV2-ELISA were confirmed. The coating concentration of the virus was 1ug/aperture /100μl , serum sample dilution was 1:80 and secondary antibody(HRP-SPA) dilution was 1:30000. Special experimentation showed that there were no interreaction to classical swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus. Comparing with imported PCV2-ELISA kit (IDXXX), the total accordance rate was 88.89% (40/45) , the positive accordance rate was 92.86% (13/14) and the negative accordance rate was 87.1 % (27/31). (5) 309 sera samples of 17 intensive swine farms were examined with the ELISA, which were collected from Fuzhou, Nanping, Sanming, Zhangzhou, Putian.it's show that the total positive rate was 29.13% (90/309).CONCLUSION: (l)The PCV2 PCR we developed was more expeditious, repetitious and sensitive than virus isolation, it was suitable for diagnosing PCV2 antigen. (2) PCV2 showed better growth and proliferation, and higher positive rate of virus isolation with treatment of D(+)-glucosamine.(3) Method4 was the best method for PCV2 purification. (4) The indirect enzyme-linked immunosorbent assay (ELISA) that we found was optimized, specific, repetitious and stable, it can be used for PCV2 sero-epidemiological investigation. |
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