基于EVM009基因的鼠痘病毒荧光定量检测方法建立OA

To establish a rapid assay for the detection of Ectromelia virus(ECTV),a real-time quantitative PCR method targeting the EVM009 gene of ECTV was developed in this study.Based on the whole gene sequence of ECTV,four pairs of qPCR primers were designed and evaluated for their specificity and sensitivity,and a pair of re-al-time quantitative PCR primers with high specificity and sensitivity targeting the EVM009 gene of ECTV were obtained by screening.A standard curve was constructed based on the pCAGGS-EVM009 eukaryotic expression plasmid.The standard curve was y=-3.509 9x+41.30,linear correlation coefficient R2=0.999 2.The amplifica-tion efficiency reached 92.7％,the lowest detection limit of the recombinant plasmid standard was 4.91 ×1010 cop-ies/μL,and the coefficients of variation of the repeated tests within and between the groups were all less than 2％,it could only effectively detect ECTV,with no cross-reactivity with VACV,GPTV,SPPV,ORFV,or LSDV.This shows that the qPCR method is specific,sensitive,and reproducible and can be used for the determination of viral load in pathological tissues of ECTV-infected mice.