绵羊痘病毒TaqMan-MGB荧光定量PCR快速检测方法的建立

Establishment of TaqMan-MGB real-time fluorescent PCR assay for rapid detection of sheep pox virus

为建立快速检测绵羊痘病毒(SPPV)快速检测方法基因的方法,本研究参照SPPV ORF068基因设计并合成一对特异性引物和一条MGB探针,经条件优化,建立了SPPV TaqMan-MGB荧光定量PCR快速检测方法。结果显示,该方法可以特异性检测SPPV,而对羊口疮病毒和猪水疱病病毒等扩增结果均为阴性;而且检出下限为10拷贝/μL;组内组间重复性试验的变异系数均小于3%。上述结果显示本研究建立的TaqMan-MGB qPCR方法特异性强、灵敏度高、重复性好,能够对SPPV进行快速准确的检测。

In order to establish a rapid method for the detection of sheep pox virus （SPPV）, the real-time PCR was developed with a pair of specific primers and one TaqMan-MGB probe targeting the ORF068 gene for SPPV detection. Then, the reaction conditions were optimized with the recombinant plasmid containing the ORF068 gene fragment for the standard curve establishment. The results showed that the method was specifically amplified target gene from SPPV, and had no cross-amplification of the off virus and swine vesicular disease virus. The minimum detectable limit was l0 copies/p,L of the virus. The variation coefficient of intro- and inter assay were both less than 3%. The method had high sensitivity, specificity and reproducibility, which could be used to application in clinical detection of SPPV infection.