Theresearchabout Traditional Weakening Test And Rapid Immunology Detection Method Of PEDV

A technology system for rapid detecting pathogen of porcine epidemic diarrhea?PED?was drawn up using the principle and technology of colloidal gold immunochromatographic assay.This study focused on developing and evaluatingthe method for rapid detection of Porcine epidemic diarrhea virus and the results were reported as follows.The PEDV South China isolate strain named as CHYJ130330 was continuously passaged from F150 to F214.TCID₅₀ is measured every 10 generations for the collected virus solution.As a result,it was observed that the TCID₅₀ was in a downward trend as a whole.Conducting the PEDV vaccinated on 3 day old piglets,the results showed that the incidence rates of the virulentvirus group,the F150 virus group,the virus F200 group and the blank control group were 100%,100%,0%,and 0%,respectively,and the mortality rate was 60%.0%,0%,0%,RT-PCR PEDV virus positive detection rate was 100%,80%,20%,0%.The results showed that the pathogenicity of the virus was reduced after subculture.The PEDV South China isolate CHYJ130330 strain was expanded with Vero cells,and PEDV was purified and concentrated by ammonium sulfate precipitation and sucrose density gradient centrifugation.Then,BALB/c mice were immunized with PEDVantigen.The splenic cells of BALB/c mice and SP2/0 cells were fused and screened in HAT culture medium.The hybridoma cell strains stably secreting mAb for PEDV were screened by indirect ELISAand11 cell strains were clonedwhich could secreted the mAb against PEDV.After inoculation in mice,the titers of 8 strains were measured at 1:1.6×10⁴,and 6 strains had better specificity.Purified ascites and high purity monoclonal antibodies were obtained by ammonium sulfate precipitation and ProteinG affinity column chromatography.The ascites of mice for PEDV were purified by bitterness-saturated ammonium sulfate method and the applicative antibodies were selected by Double antibody sandwich ELISA andcolloidal gold immunochromatograohic assay.The specificity,purity and subclass of antibodies were analysed by indirect ELISA.One of them named with 5A10 and 3A11 that was successfully used in the colloidal goldimmunochromatograohic assay.The repeatability,specificity,and sensitivity of the assay were evaluated.Clinical samples were detection by this assay and compared with RT-PCR.The limit of detection was 7.8×10³TCID₅₀/mL.The specific experiments showed thisassay had no cross reaction with PRV,CSFV,PRRSV,TGEV.The assay established in this study was reproducible,specific and sensitive.115 clinical samples were detected.The coincidence rate with RT-PCR was 93%.This assay establish by us provides a basis for the development of mature PEDV rapid detection colloidal gold test strip products.