区别伪狂犬病病毒野毒株和gE-疫苗株的gE-MGB-TaqMan PCR方法的建立

本实验通过设计针对伪狂犬病病毒gE基因的引物和MGB(Minor groove binder oligodeoxynucleotide conjugate,MGB-ODN)TaqMan探针,结合ABI PE7700荧光定量PCR仪器系统,建立了一种能区别伪狂犬病病毒野毒株和gE-疫苗株的快速检测方法gE-MGB-TaqMan PCR。实验表明,该方法可检测出最低43拷贝的gE基因和10~4倍稀释的伪狂犬病病毒Fa株DNA,与微量血清中和结合MTT比色法相比,灵敏度和特异性一致,检测时间仅为后者的1/10,操作比后者更为简单。特异性和重复性试验表明:gE-MGB-TaqMan PCR特异性和重复性好。该方法以闭管的模式操作,减少了各步骤污染的可能性,整个检测少于3 h。

A gE-MGB-TaqMan real-time PCR to differentiate pseudorabies virus gE gene detected vaccine strains from wild-type strains

A fluorogenic quantitative PCR was established for detecting and differentiating PRV wild-type strains from vaccine strains depleted of gE gene.The method used a pair of primers specific for gE gene and an internal dual-labeled fluorogenic probe of PRV gE gene based on the ABI PE7700 detection system.The assay was able to detect 43 copies of gE gene and 10~(-4)dilution of PRV Fa strain DNA,with a sensitivity and specificity comparable to that of micro sera neutralization combined with MTT detection.However the PCR method took only 1/10 of the time reqired by MTT and can be performed in less than 3 hours.Also the simplicity of the assay minimized the risk of contamination of subsequent procedures.