Development and Application of TaqMan Fluorescence Quantitative PCR Assay for Detecting Classical Swine Fever Virus

According to the conserved sequence of 5′nontranslated region(5′NTR)of classical swine fever virus(CSFV)in GenBank,apair of primers and a TaqMan probe were designed.The recombinant plasmid pGEM-CSFV containing the complete gene of CSFV was as a standard control to establish fluorescence quantitative PCR standard curve.Specificity,sensitivity and reproducibility of the method were determined.CSFV load in plasma was measured at different time points after experimental infection of CSFV in pigs.The results showed that standard curve established had a good linear relationship between threshold cycle and template concentration,R2=0.999.The assay had high sensitivity and could detect 1×101copies/μL.The specificity of the method was high,there was no cross reaction between CSFV and other pathogens containing porcine circovirus 2,porcine parvovirus,porcine pseudorabies virus and porcine reproductive and respiratory syndrome virus.The method showed good reproducibility,there was a coefficient of variations less than 2%for both inter-assay and intra-assay.CSFV load in plasma increased to peak value at 7days after experimental infection of CSFV in swine and gradually decreased later.These results indicated that the developed TaqMan fluorescence quantitative PCR assay had the advantages of good specificity,sensitivity and reproducibility.It provided a basis for quantification analysis and clinical diagnosis of CSFV.