Establishment and application of TaqMan real-time fluorescent quantitative PCR for detecting porcine circovirus typeII

【Objective】 The purpose of this study was to establish a TaqMan real-time fluorescence quantitative PCR method which can detect porcine circovirus typeⅡ quickly and accurately in clinic.【Method】 According to the published complete genome sequence of PCV-2 isolate SX-1 in GenBank,a TaqMan probe and a pair of primers were designed and synthesized for the conversed open reading frame 2(ORF2) of PCV-2.After the gene fragment was amplified using designed primers,the amplified product was identified.Recovery of PCR amplified products,connection of pMD19-T vector,transformation of E.coli DH5α,and coating of Amp+agar plates were conducted and the positive clones were picked for PCR and sequencing identification.Then the plasmids were extracted as the standard plasmid of fluorescent quantitative PCR.The standard plasmid was used as a quantitative template to establish the TaqMan real-time fluorescence quantitative PCR method for PCV-2 detecting,and sensitivity,stability and specificity of the method were evaluated.56 clinical samples were detected with the established method and their results were compared with conventional PCR.【Result】 The TaqMan real-time fluorescence quantitative PCR method was established and the correlation coefficient(R2) and the slope of the standard curve were 0.998 and-3.383 respectively,which showed a good linear relationship.Repeatability tests showed that the threshold cycle of the method had very good stability with the coefficient of variations(CV) between assays within 0.86%-1.02%.Results of sensitivity tests showed that the detection limit was 5.06 copies/μL.Specificity tests showed that the detection results of porcine circovirus typeⅠ,pseudorabies virus,porcine reproductive and respiratory syndrome virus,and class swine fever virus samples were negative while that of the standard PCV-2 sample was positive,indicating a good specificity.In clinical samples tests,detection rate of established fluorescence PCR was 14.3% higher than that of conventional PCR.【Conclusion】 This study established a TaqMan real-time fluorescence quantitative PCR method which can detect PCV-2 and be used to detect the clinical PCV-2 samples.