Isolation And Identification Of PEDV Strain With Establishment Of Antibody ELISA Detection Methods

Porcine epidemic diarrhea(PED)is an acute,highly pathogenic intestinal infectious disease caused by Porcine epidemic diarrhea virus(PEDV),which mainly harms suckling piglets.The clinical manifestations are watery diarrhea,vomiting and dehydration.It is a more effective prevention method for suckling piglets to obtain passive immune protection through maternal antibodies(secreted IgA)in milk,and detection of antibodies in milk is of great significance for the prevention and control of PED.In this study,a strain of PEDV was isolated and identified from the clinical test materials.The N protein derived from the strain was expressed in HEK-293 suspension cells,and the indirect ELISA methods were established based on the recombinant N antigen for the detection of PEDV serum IgG and milk IgA antibodies,respectively.The ELISA method provides support for the prevention of PEDV.Related research results are as follows: 1.Isolation and identification of PEDV-JX18 strainIn 2018,the small intestine tissue suspected of being infected with PEDV was from a pig farm in Jiangxi Province.It was minced and ground,and the ground tissue was soaked with cell culture medium overnight to obtain the virus solution.The virus was isolated in Vero cells and observed on Vero cells appeared the cytopathic effects until the fifth generation.Cultures were identified by RTPCR,IFA,transmission electron microscopy and ultrathin cell sections,and a field Porcine epidemic diarrhea virus was isolated and named PEDV-JX18 strain.2.Establishment of an indirect ELISA method for PEDV serum IgGThe N gene of PEDV-JX18 strain was cloned into pcDNA3.4(+)plasmid,transiently transfected with HEK-293 suspension culture cells and purified to get N protein,which was used as the coating antigen for the indirect ELISA method for the development of PEDV serum IgG antibody.The best reaction conditions are determined by optimizing dosage and incubation time of each component.The antigen coating amount is 2 mg / mL,the optimal serum dilution is 1:80,the incubation time is 37 ? for 30 min,and the goat anti-pig IgG-HRP dilution concentration is 1: 4000,incubation time is 37 ? for 1.5 h,after that the duration of reaction with TMB was 37 ? for 15 min.Using 40 PEDV positive sera and 178 negative sera samples to evaluate the indirect ELISA method,SPSS 17.0 software was used to draw ROC curve to judge the results,and the critical value was 0.585,the sensitivity was 100 %,and the specificity was 96.7 %.Repeatability test results show that the coefficients of variation are all less than 10 %.The result indicated that the established indirect ELISA method for detecting PEDV serum IgG has excellent stability and repeatability.3.Establishment of PEDV milk IgA indirect ELISA methodThe recombinant PEDV N protein expressed by HEK-293 cells was used as the coating antigen to establish an indirect ELISA method for detecting IgA antibodies in PEDV milk.The best reaction conditions are determined by optimizing dosage and incubation time of each component.The best antigen coating amount is 1 mg/mL,the best milk dilution factor is 1: 2,the milk incubation time is 37 ? for 30 min,and the goat anti-pig IgA-HRP dilution concentration is 1: 2000,the incubation time is 37 ? for 1 h,after that the duration of reaction with TMB was 37 ? for 10 min.31 milk positive samples and 25 negative milk samples stored in the laboratory These milk samples,including 31 positives and 25 negatives,were stored in our laboratory,which was analyzed to draw ROC curves by SPSS 17.0 software..The critical value was 0.653,the sensitivity was 100 %,and the specificity was 96 %.Repeatability test results show that the coefficients of variation are all less than 10 %.The established indirect ELISA method for detecting PEDV milk IgA has good stability and repeatability.In this study,a PEDV field strain was successfully isolated and named PEDV-JX18 strain.The N protein was successfully expressed in HEK-293 cells and used as a coating antigen to establish the indirect ELISA method for detecting the PEDV IgG antibody in serum and IgA antibody in milk,which provides technical support for the clinical prevention of PEDV.