| Porcine epidemic diarrhea(PED),Transmissible dastroenteritis(TGE)and Porcine rotavirus disease(RVD)are common viral diarrhea diseases in pig farms,which are caused by Porcine epidemic diarrhea virus(PEDV),Transmissible gastroenteritis virus(TGEV)and Porcine rotavirus(RV),respectively,bringing huge economic losses.The three kinds of viral diarrhea diseases,with similar transmission routes and clinical symptoms,cause watery diarrhea,vomiting,dehydration and high mortality of new-born piglet.The epidemiology and pathological autopsy of the three diseases are extremely similar,however,there is no common antigenicity and interaction in the three pathogenies.The new-born piglets are grow slowly and have high mortality because of mixed infection and other bacterial infection.At present,the conventional diagnostic methods,including virus separation and serological diagnosis method and so on,can't meet the need of the clinical diagnosis.However,the multiple polymerase chain reaction(PCR)is more suitable for the rapid diagnosis of mixed infection with of high sensitivity and the characteristics of testing numerous pathogens at one time.It has been found that immune pigs appear the phenomenon of PED.To testing the immune level of PEDV in pig farms of Shandong province,we detected the antibody of immune pigs and established the prokaryotic expression of antigen epitope of S gene as well as we established the indirect ELISA method of s90 protein.The results of the study are as follows: 1.The establish of multiple PCR method of PEDV-TGEV-RVIn this study,the multiple PCR method of PEDV,TGEV and RV was established and whose specific primers were designed according to their conservative gene of M,N and VP7,with the length of PCR product was 311 bp,763bp and 516 bp.We successfully established the multiple PCR method of the three common diseases in pig farms.With the sensitivity and specificity test done,the highest sensitive of PEDV,TGEV and RV is 30 pg,18pg and 42 pg,respectively;and the specificity of this method is well and can be used to clinical test by using PCR amplification other common viruses.We totally tested 412 samples from Jinan,Taian,Laiwu and son on in Shandong province.The results showed that the positive rate of PEDV,TGEV and RV is 22.3%,7.28% and1.46%,respectively;PEDV+TGEV?PEDV+RV and TGEV+RV the double infection rates is 5.34%,1.94% and 2.91% while the triple infection positive rate is 4.85%.In addition,the single RT-PCR method being reported was used to test our samples and the coincidence rate was as high as100%,indicating that the multiple PCR method was sensitivity and specificity and can be used to test PEDV,TGEV and RV in the clinical.2.The prokaryotic expression of S90 of PEDVIn this study,one pair primer was designed,which was used to amplify the antigen epitope of S gene of PEDV in Shandong province.The PCR product was then connected to the pET-32a(+)vector,constructing the recombinant expression plasmid,which was last imported into E.coli BL21(DE3).The stable expression system was constructed by testing the expression time,the quantity of inductor and so on.The results showed that the best expression conditions are as follows: IPTG is 1m M/mL,37?,220 rpm and the largest quantity is at 6 hours.The high purity of target protein was obtained after purification,and Western blot showed that the expression of recombinant protein S90 can specifically react with positive serum of PEDV and not react with other positive serum of pig disease.These results suggested that the protein has a certain reactionogenicity.3.The establishment and application of indirect ELISA method of PEDVIn this study,the purification of prokaryotic expression S 90 was used as the envelope antigen,and then we established the indirect ELISA method to detect the antibody of PEDV.The optimal concentration of protein is 2?g/mL with incubation overnight at 4?and the optimal coating buffer is 5% skim milk powder.In addition,the optimal concentration of pig serum and the secondary antibody is 1:100 and 1:4000 at 37? for 2h and 1.5h,respectively.The optimal terminal time of TMB substrate is 15 min and the cut-off value of positive is 0.204.We totally detect 120 pigs from two non-immune pig farms of Laiwu of Shandong province.The overall antibody detection was 54.15%,among which the positive antibody rate of sows and the breeding pigs was 60% and 48.3%,respectively.These results suggested that there is serious wild poisonous phenomenon because of the low positive rate of antibody detection.In addtion,we detected and traced the antibody level of 195 pigs in three pig farms of Jinan of Shandong province.The positive antibody rate of PEDV in detective serum was 70.3%,among which the positive PEDV antibody rate of sows and the breeding pigs was 64.8% and 76.7%,respectively.Moreover,pigs immuned with PEDV vaccine 7d antibody began to rise and achieved the highest at 28 days according to tracing the rulses of antibody level of pigs. |
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