Development of Taqman real time fluorescent quantitative PCR for porcine circovirus type 2

Porcine circovirus type 2 (PCV2) is a pathogen causing postweaning multisystemic wasting syndrome (PMWS), immune suppression, and skin nephrotic syndrome of piglets. To construct specific method of detecting PCV2 nucleic acid basing on Taqman probes, PCV2 genomic DNA was used as a template for polymerase chain reaction (PCR). The specific primers of PCV2, open reading frame 1 (ORF1), and probes were designed and synthesized according to the gene sequence in GenBank. Then, the target fragment was amplified and cloned into a pMD18-T vector. The cyclic conditions of quantitative PCR were optimized by a template with a positive plasmid standard. The standard curve of real time fluorescent quantitative PCR for PCV2 was established and the sensitivity, repeatability, and specificity of the method were verified by three repeatability tests. Results showed that a positive plasmid was successfully constructed. The optimal concentration of primers and probe was 0.2 μmol·L-1, and the minimum detection threshold was 8.828×106 copies·L-1. The linear range of detection was 1013-106 copies·L-1. This method had no cross reaction of related diseases, a low coefficient of variation, and well repeatability between testing of every batch. From this study then, the high sensitivity and specific real-time quantitative PCR for PCV2 could be used as a tool for rapidly diagnosing PCV2 infectious diseases.