Development And Application Of Fluorescent Quantitative PCR Assay By TaqMan Probe Approach For PCV2 Detection

Porcine circovirus type 2(PCV2) was considered being associated with Postweaning Multisystemic Wasting Syndrome (PMWS), which was broken out all over the world in recent years.PMWS was clinically characterized by wasting and multisystemic pathologic damnification.furthermore,the infection of PCV2 can induced immunosuppression which can lead to the sequential inflection of other pathogens.The wide prevalence and spreading of the disease brought out serious threat to pig industry and huge loss in many nations and regions in the world. In the present study,a fluorescent quantitative PCR assay by TaqMan probe approach for PCV2 detection was established and was used for PCV2 detection in blood among various pigs in a hoggery in Guangdong province of China.1.PCV2 ORF2 gene cloning A pair of primers was designed according to the PCV2 gene sequence in GenBank. The ORF2 gene, a fragment of 702bp , was amplified by PCR from PK-15 cells inoculated with PCV2 samples. The PCR products were cloned into pMD 18-T Easy vector and identified by KpnI and HindⅢdigestion, the recombinant plasmids named pMD-ORF2 were obtained.2.Establishment of FQ-PCR for PCV2 detection A pair of primers and a TaqMan probe were designed according to the published ORF2 gene sequences of PCV2 in GenBank. The FQ-PCR assay was carried out by quantitative concentration of serial 10 fold dilutions of pMD-ORF2 DNA by optimizing circulation parameters. A standard curve was achieved and the result showed the sensitivity of this method was 1.0×100copy/μL and the linear relation was excellent. Meanwhile, the final measure value on initiative concentration of 1.0×106,1.0×105,1.0×104copy/μL DNA was 1.001×106,0.935×105 and 0.987×104copy/μL and the coefficient of variation was 2.527%,2.067% and 2.092%, respectively. Furthermore, we found the sensitivity of FQ-PCR was roughly equal to that of nPCR and was prior to that of PCR by detecting the positive field samples.3.Detection of PCV2 in various pig′s blood The results of detection of PCV2 in various pig′s blood different groups showed that the PCV2 inflection was consisted in all...