Establishment Of Double RT-PCR Detection Method For Pig Diarrhea And Antigenic Inactivation And Immune Effect Evaluation Of PED

Porcine epidemic diarrhea is a kind of acute,highly contactable intestinal infectious disease that can be caused by swine epidemic diarrhea virus in pigs that at different stages of age.The primary clinical features are diarrhea,vomiting,dehydration and high mortality rate of suckling pigs,especially 3-10 days piglets.Since the discovery of it in 1971,it has become one of the major viral infectious diseases that seriously affect the pig industry.Therefore,it is very important to do a good job of prevention and control of porcine epidemic diarrhea.Through research and analysis of the diseased materials submitted by the JiLin Agricultural Science and Technology University in recent years,there are two kinds of viral diseases that cause diarrhea in pigs in Jilin region,epidemic diarrhea(PED)and infectious gastroenteritis(TGE),and there are cross infections in both diseases at most circumstances.Therefore,based on the PED,this study establishes a dual RT-PCR method for the detection of PEDV and TGEV.Firstly,the positive materials of the simple infection of PED,the double infection of PED and TGE were screened by colloidal gold kit and conventional RT-PCR method;based on the comparison analysis of the sequence published by GeneBank,design the primers of PEDV and TGEV.The lengths of the target genes were 832 bp and 223 bp.The double RT-PCR method was repeated several times.The optimal conditions for the final reaction were Premix Taq DNA enzyme 12.5 ?L,PEDV upstream primer 1 ?L,PEDV downstream primer1 ?L,TGEV upstream primer 1 ?L,TGEV downstream primer 1 ?L,cDNA template 2.5 ?L,sterilized double-distilled water 6 ?L.The total system was 25 ?L.The best amplification procedure was: 94°C for 2 minutes;94°C for 30 seconds,54°C for 30 seconds,72°C for 1 minute,35 cycles;72°C for 10 minutes.The results of the test showed that the dual RT-PCR of PEDV and TGEV has great specificity.It can be used as a rapid clinical test for PED and TGE.Pig epidemic diarrhea and transmissible gastroenteritis and other viral diseases causing diarrhea were detected in pigs at a pig farm in Jilin through the colloidal gold detection and the double RT-PCR assay established above.The results showed that just a simple infection with porcine epidemic diarrhea virus(PEDV),which eliminate mixed infection with other viral diarrhea.Collect the intestinal tissues and lymph nodes from diseased pigs diagnosed as PEDV,and the inactivated tissue vaccine is prepared by conventional methods: after inactivation with 0.4% formaldehyde solution,double-antibody treatment,sterility test,and safety test,the inactivated tissue vaccine were prepared.It was used to immunize 45 healthy pigs at the experimental farm,including 18 sows,18 piglets,and 9 replacement gilts.The ELISA method was used to monitor the antibody level before and after the use of inactivated tissue vaccines,and compare the immune effects of two conventional vaccines,which are commercially available in the control group.Explore the best immunization program for preventing this disease and provide technical support for control of epidemic diarrhea effectively.The test results showed that the measured antibody levels increased significantly before and after the use of tissue-inactivated vaccines.Compared with the other two commercially available vaccines,the mean increase was larger,but the dispersion was higher.Overall it works well.