Development of TaqMan probe-based fluorescent quantitative PCR method for porcine cir-covirus type 2

Objective To develop a TaqMan probe-based fluorescent quantitative PCR method for porcine circovirus type 2(PCV2).Methods Two pairs of specific primers and TaqMan probe were designed according to the relatively conserved sequence of PCV2 ORF2 for preparation of standard for recombinant plasmid.The reaction system and condition were optimized,based on which the standard curve was plotted,and a TaqMan probe-based fluorescent quantitative PCR method was developed and verified for sensitivity,specificity and reproducibility.Fifty clinical samples were determined by the developed method,and the results were compared with those by routine PCR method.Results PCR and sequencing proved that the standard for recombinant plasmid was constructed correctly.The Ct value of plotted standard curve showed good linearity to the log of copy number of template,with a R2 value of 0.993 85.The sensitivity of developed method was 6 × 101 copies/μl,which was two orders of magnitude higher than that of routine PCR method.No amplification curves were obtained from porcine reproductive and respiratory syndrome virus(PRRSV),porcine pseudorabies virus or attenuated lapinized classical swine fever virus.The variation coefficients of determination results of three PCV2-positive templates by the developed method were 0.07% ～ 0.50%.The positive rate of clinical samples by the developed method(64%) was significantly higher than that by routine PCR method(44%)(P 0.05).Conclusion The developed TaqMan probe-based fluorescent quantitative PCR method showed high sensitivity,specificity and reproducibility,which was suitable for rapid detection of trace PCV2 in clinical samples as well as study on tissue tropism and cell culture characteristics of PCV2.