Development And Application Of A Competitive Quantitative Assay For Monitoring Porcine Circovirus DNA

A multiplex polymerase chain reaction was developed to simultamneous detect type 1 and type 2 of porcine circovirus. The homology of the full sequences of PCV reported and registered in Genebank of different strains of PCV-1 and PCV-2 was respectively and compared with each other. Two sets of specific primers of PCV were designed. PCV can be amplified 938bp, PCV-2 can be amplified 490bp.The designed primers were used to PCR reaction. The result showed that the design of the two sets of primers was right. Then the optimal condition of PCR for PCV were determined.Competitive quantitative PCR (cPCR) assay was developed for monitoring and detecting porcine circovirus (PCV-2) DNA. Using PCR assay, the cPCR was based on competitive co-amplification of a 490-bp region of the PCV-2 ORF2 gene, with a known concentration of competitor DNA, which produced a 692-bp fragment. The cPCR was validated by quantification of a known amount of competitor PCV DNA with one series of dilutions of target DNA. Using the regression equation, we protracted the standard curve and affirmed the quantity of the competitive template. According to the different of the IOD between the products of competitor and target, introduce Lc/Lt as the adjust coefficient. After all parameter regressed, got the concentration formulae that based on the quantity of the samples and the IOD of the PCR products.Used this technique to determine PCV genome copy numbers in infected cells. Furthermore, we measured PCV DNA loads in clinical samples and found PCV-2 DNA content in the piglet with PMWS is higher than 1.0145 x 108copies/ml in lung, 0.8 x 107copies/ml in blood, and 9.698 x 108copies/ml in lymph. Our research might provide some hints to the study of the future work.