Development And Application Of A QPCR For PEDV And An Indirect ELISA For Detection Of Antibodies Against Porcine Epidemic Diarrhea Disease Virus

Porcine epidemic diarrhea(PED) is a highly contagious intestinal disease of pigs caused byporcine epidemic diarrhea virus(PEDV). The main characteristic of PEDV was vomiting,diarrhea and loss of appetite in a variety of age pigs, especially piglets. But in recent years,the outbreak of diarrhea which was caused by PEDV was happened in ShandongProvince,and high mortality was appeared in this outbreak. In order to effectively control thedisease, This study established a qPCR method to detect PEDV and indirect ELISA method todetect antibodies of PEDV. The study is divided into three parts:1. The establishment of a qPCR for PEDVAccording PEDV M gene sequence of DZ12-3, the study synthesized Taqman probe andprimer sequences, then I establish an absolute quantitative PCR method for detection ofPEDV. The standard curve equation was Y=-2.146X+22.769.The Slope,amplificationefficiency and the correlation coefficient were respectively-2.146, E=10-1/斜率-1=10-1/-2.146-1=192.433%and R2=0.994. The study show that the method had goodspecificity and reproducibility, high sensitivity, minimum detectable could be10copies/μL..Forty clinical symptoms of diarrhea disease materials from Shandong were tested by thefluorescent PCR for PEDV. It was showed that30materials were positive and the detectionrate was75%.2. The prokaryotic expression for the N gene of PEDVShandong epidemic strain DZ12-3was used as a template in this study,and the completesequence of N gene was amplified through designing specific primers.The recombinantplasmid was built by connecting the N gene with pET-30a,and imported into the E.coliBL219(DE3). Through the exploration of the expression of time, the amount of inducer, thetemperature and other conditions, so that constructed plasmid pET30a-PEDVN had a largenumber of expression stably in BL21, and determination of the optimal conditions are the1mM/mL expression of IPTG,37℃,220r/min and that maximum amount of expression timewas6h..Then the high purity protein was obtained after passing the Ni-Agarosecolumn.Tested by Western blot,it was showed that the recombinant protein had a goodspecific reaction with the positive serum of PEDV,while not had a reaction with positive serum of other diseases. Therefore, this protein had a good reactogenicity.3. The establishment and application for PEDV of indirect ELISA methodThis study established the indirect ELISA method based on the purified N protein forPEDV antibody The optimal amount of N protein coating was750ng per hole, Optimalcoating conditions were the use of carbonate buffer (0.05mol/L, pH9.6) and packeting of4℃,12h; the subject pig serum dilution was1:100;the best antibody optimum reaction timeis37℃,1h, the best color liquid termination time15min at room temperature, positiveCut-off value of0.465.The320porcine serums by PEDV vaccine were tested PEDV antibody by the assay ofindirect ELISA, and it showed that the total antibody positive rate was62.81%, the antibodypositive rate of sow was72.2%and the positive rate of piglet was46.1%. It Showed antibodyvaccine protection rate is not high. Detected120unvaccinated PEDV vaccine sera from twofarms in Dezhou, statistics showed that the total infection rate of36.7%the antibody positiverate of sow was42.9%, the positive rate of piglet was28.0%. t The results showed thatporcine farms had generally PEDV wild virus infection, Through display of tracking andmonitoring of a pig farm after immunization of PEDV vaccine antibody, antibodies began torise after7d, reached the highest after28days, decline appeared after35d, The results showedthat the PEDV vaccine had shorter protection and poor protection.