中文摘要：

目的建立一种温州病毒（Wenzhou virus,WENV）的SYBR Green Ⅰ实时荧光PCR检测方法。方法分析WENV保守区域并设计特异引物,以特异扩增产物构建的阳性重组质粒为标准品,建立SYBR Green Ⅰ实时荧光定量PCR反应的扩增曲线和溶解曲线,获得标准曲线。结果建立的WENV的荧光定量PCR标准曲线在1×10~1~1×10~8拷贝/μl的浓度时呈现良好线性关系,其扩增相关系数为0.999,熔解曲线只出现1个特异峰,无引物二聚体,检测下限可达10拷贝。利用WENV的样本对本检测方法进行验证,结果良好,组内变异系数为1.47%。讨论本实验建立的SYBR Green Ⅰ实时荧光定量PCR检测方法灵敏度高、特异性好,可用于WENV的诊断及病原的定量分析。

英文摘要：

Objective： To establish SYBR GreenⅠreal-time PCR method for Wenzhou virus（ WENV）. Methods： The conserved fragments of RNA-dependent RNA polymerase（ rdrp） gene of WENV were analyzed. Primers were designed based on the conserved regions. The recombinant plasmid was used to establish melting and standard curves of SYBR GreenⅠreal-time PCR. Results： The generated standard curve had a wide dynamic range from 1 × 10~1 to 10~8 copies/μl,with a linear correlation of R~2= 0. 999. The melting curve analysis using SYBR GreenⅠshowed that only one specific melting peak was observed and no primer-dimers represented. The detection limit of the method was 10 copies/reaction and intra-assay variation was 1. 47%. Conclusion： The established SYBR GreenⅠreal-time PCR method has high sensitivity and strong specificity,which is suitable for rapid detection of Wenzhou virus.