Development and application of SYBR-Green I real-time quantitative PCR technique for detecting porcine parvovirus virus

According to the porcine parvovirus virus strain NADL-2(NC001718) VP2 gene sequence available in GenBank,a pair of specificity which can amplify about 431bp fragment was designed.The purified PCR products were inserted into pMD18-T vector,and then transformed to E.coli DH5α.After PCR identifying and sequencing,recombinant plasmid was extracted as a positive template to establish SYBR-GreenⅠfluorescence quantitative PCR standard curve.The method of sensitivity,reproducibility and specificity were determined.The results indicated that standard curve established by recombinant plasmid showed a good linear relationship between threshold cycle and template concentration,R2=0.997 6,the Tm was between 82.3-82.9℃ and the sensitive degree is 72.1 copies per μL,and the quantitative PCR was high reproducibility and more specific than that of traditional PCR.A SYBR Green Ⅰ fluorescent quantitative PCR assay for detecting VP2 gene of PPV was developed and provided a basis of the early,rapid detection and quantitative analyzation for the infect degree of PEDV.