马疱疹病毒1型SYBR GreenⅠ荧光定量PCR检测方法的建立

Establishment of the SYBR GreenⅠreal-time PCR for detection of equine herpesvirus type 1

为建立马疱疹病毒Ⅰ型(EHV-1)的检测方法,本研究以EHV-1 gB基因的一段保守区域(1 207 bp～1 509 bp)作为检测的目的片段设计引物,通过对其反应条件的优化,建立了特异性检测EHV-1的SYBR GreenⅠ荧光定量PCR方法。实验结果表明:该方法检测目的基因的灵敏度下限为10拷贝/μL,比常规PCR方法高100倍;与马疱疹病毒4型(EHV-4)及其他马传染病病原体无交叉反应;组内及组间的变异系数均小于2%。该方法检测速度快及高敏感性的特点为马鼻肺炎的防制提供了有力保障,同时也为进一步开展马鼻肺炎相关的研究提供了有效的辅助检测方法技术。

Equine herpesvirus-1 （EHV-1） is one of the pathogens which causes equine rhinopneumonitis in horses, l＇o establish a method for EHV-I detection, the SYBR Green I real-time PCR was developed with primers targeting the conserved sequence of gB gene of EHV-1. The limit detection of real-time PCR was about 10 copies for EHV-1 which was 100 times higherthan normal PCR, and no cross-reaction to equine herpesvirus-4, equine influenza virus, equine artcritis virus and Japanese encephalitis virus. The repeatability tests indicated that the coefficient of variation were less than 2% in both intra-assay and inter-assay. These results demonstrated the established real-time PCR was suitable for rapid detection of EHV-1 and for the lhrthcrstudy for EHV-1.