Development Of Multiplex PCR For Diagnosing Five Reproductive Failure Diseases Of Porcine

A multiplex polymerase chain reaction was established for differential diagnosis five viral pathogens of porcine. According to the conservative sequences, registered in GenBank, of different strains of NS1 for PPV, ORF2 for PCV2, NS1 for JEV, M gene for PRRSV and gE gene for PRV, 5 pairs primers were designed and analyzed with Primer Premier 5.0 and Oligo 6.0 softwares. Different length fragments would be amplified with the special primers, 750bp for PPV, 494bp for PCV2, 475bp for JEV, 363bp for PRRSV(American genetype) and 178bp for PRV, respectively. Through optimizing the major factors (e. g., PCR recation Buffer, Annealing temperature, dosage of Taq DNA polyperase, primers concentration, Mg²+ concentration, etc. ) which influence the amplification efficacy of PCR, we got initially five single PCRs to detect those five diseases sensitively. At the same time, A comparative study was performed with three different template DNA extract ways(I. e, by kit, by proteinase K, by Phol-Chl) of PCV2, we confirm the best template extract mathod (extracting by Kit). Further, we went on to optimize the dosage of Taq DNA polyperase, primersconcentration and proportion, Mg²+ concentration, etc. Two reactions had been established at the same reaction procedure finally. One is amplification for PPV-JEV, and another is for PCV2-PRRSV-PRV. The specificity of our assay was verified by performing the Muiti-PCR with DNA from PRV-SA215 strain, PCV1 and the nucleinic acid extract of lymphatica of medical fitness pigs. None of these virus and sample of tissue gave the corresponding amplication product. Therefore, the primers described here proved to be specific under the conditions assayed. Our Multi-PCR assay could detect as few as 40.7TCID₅0PPV, 15.5TCID₅0JEV, 389TCID₅0 PRRSV, 245TCID₅0 PRV and 10^-4DNA extract of PCV2. Using this Multi-PCR, we detected 30 diseased materials collected from infected pig-farms of Sichuan, Chongqing, Guangxi, Guangdong and Fujian province/Municipality, The final result, together, compared with those obtained in Single-PCR, the average positive coincidence rate reach to 93.3%, indicate the procedure is a highly sensitivities and specific mathod for detecting these five pathogens in pig farm outbreaks.