Construction of standard positive template of the fluorescent quantitative PCR assay for detection of swine fever virus.

5′UTR of swine fever virus(SFV) was amplified by RT-PCR and cloned into pMD-18 simple T vector to construct a recombinant plasmid pSF,which was used as standard positive template for the fluorescent quantitative PCR(FQ-PCR) to detect SFV.The pSF was identified by PCR and DNA sequence analysis.The plasmid was stable after 6 months storage at-20℃.When the plasmids DNA of 1.0×109,1.0×107 and 1.0×105 copies/2 μl were used as template,the largest variation coefficient of Ct value was 1.55%,and the FQ-PCR provides a broad linear range from 1×102 to 1×1011 copies of DNA per reaction.The pSF was specific and stable for the FQ-PCR.The method can be used for the clinical diagnosis.