| Viral myocarditis,a highly contagious disease,can infect a variety of animals and people and cause encephalitis,myocarditis or myocardial inflammation.Pigs are susceptible hosts of EMCV.The infection of EMCV could cause high mortality of suckling piglets and reproductive disorder of pregnant sows,and cause great economic losses to the pig industry.Serological investigation showed that the seroprevealence of EMCV reached more than 50%in the pigs of some scaled farms that were not immunized with EMCV vaccine.The investigation indicates that EMCV infection was common among pig population and it is necessary to detect and monitor the epidemic of EMCV.For the investigation and analysis of EMCV epidemiology,we estibalished and optimized an enzyme-linked immuno sorbent assay(ELISA)for the detection of EMCV specific antibody.Firstly,a DNA sequence containing VP1 antigen according to referenced sequence in GenBank was cloned and transformed into BL21 competent cells to induce the expression of protein.The objective protein was purfied and coated to the wells of ELSIA plates.Finally,the diagnostic method of indirect ELISA for detection of EMCV antibody was obtained.We then optimized some indicators of the indirect ELISA method,and the optimized conditions were used as follows: carbonic acid buffer with pH 9.6 was used as the coated buffer,1mg/ml puridied protein was coated to the wells under 4 ℃ for 12 h,and then the optimal blocking condition is confined with 5% milk in 37 ℃ incubator for 1 h.The serum samples were1:100 diluted and incubated under 37 ℃ for 0.5h.The enzyme labeled antibody is 1:1000dilution,and incubated under 37 ℃ for 1.5h.TMB staining time is optimized as 20 min at room temperature.The OD values were obtained using microplate reader with TG-ROC software.The serum sample with an OD value that greater than 0.5 is identified as antibody positive of EMCV,an OD value that less than 0.4 is identified as negative sample,while a sample with the OD value between 0.4-0.5 is identified as a suspicious sample.We applied the method to detect 2016 serum samples collected from Shandong province.The results showed that 984 of them were positive for EMCV antibody,and the positive rate was 46.72%;while 985 samples were negative for EMCV antibody,and the negative rate was46.77%;a total of 137 samples were identified as suspicious samples,and the suspicious rateis 6.51%.The results indicated that we are faced with a severe status of EMCV infection among the pigs in Shandong Province.This study provides an effective way for the epidemiological surveillance and diagnosis of EMCV infection in Shandong Province.Moreover,the ELISA method can be used to investigate the infection status of EMCV among pigs. |
