Development and application of a SYBR GreenI real-time fluorescent quantitative PCR method for detection of PPV

【Objective】 The study developed a SYBR GreenⅠ real-time fluorescent quantitative PCR which can detect porcine parvovirus(PPV) quickly and specially.【Method】 According to protein 2(VP2) nucleotide sequences of porcine parvovirus(PPV) published in GenBank,a pair of primer was designed.The VP2 gene was amplified with traditional PCR.The PCR product was cloned into pGEM-T Easy vector and sequenced.A real-time fluorescent quantitative PCR was established by optimizing the SYBR GreenⅠ's concentration,primers concentration and Mg2+ concentration.The positive recombinant plasmid was used as quantitative template to generate standard curve and melt curve.Sensitivity assay,reproducibility of the assay and specificity assay were determined.Clinical 60 suspected PPV disease materials were detected by established SYBR GreenⅠ real-time fluorescent quantitative PCR method and compared with HA and the result of the conventional PCR testing methods.【Result】 The background response and Ct is minimum,the amplification efficiency is maximum when the Mg2+ concentration is 4.5 mol/L,SYBR GreenⅠ concentration is 20 μmol/L,primer concentration is 25 μmol/L in 25 μL amplification system.The real-time fluorescent quantitative PCR method can specificly and quantitatively detect porcine parvovirus.The detection limit of real-time PCR for PPV was 20 TCID50/mL.The detection rate is 13.3% higher than the conventional PCR in the clinical.【Conclusion】 A SYBR GreenⅠ real-time fluorescent quantitative PCR to detect VP2 gene of PPV was developed on the basis of early and rapid detection and analysis of the infect degree of PPV quantitatively.