荧光定量PCR技术用于快速检测水痘-带状疱疹病毒的研究  

Research on fluorescent quantitative TaqMan-based PCR method for rapid detection of Varicella-zoster virus

作　　者：黄世旺[1] 方叶珍[1] 卢亦愚[2] 李剑[1] 徐丹戈[1] 蒋雪凤[1] 倪志敏[1] 

机构地区：[1]杭州市江干区疾病预防控制中心,杭州310004 [2]浙江省疾病预防控制中心,杭州310051

出　　处：《中国卫生检验杂志》2012年第12期2898-2900,共3页Chinese Journal of Health Laboratory Technology

摘　　要：目的:建立特异、灵敏、高效的TaqMan荧光定量PCR方法用于水痘-带状疱疹病毒(VZV)的快速检测。方法:从GenBank下载几十株VZV基因序列进行同源性比对,选取高度保守区ORF62(编码即刻早期蛋白IE62)设计荧光定量PCR引物和TaqMan探针,并对反应体系与反应条件进行优化,同时验证方法的特异性、敏感性和重复性。结果:本方法对VZV的检测具有高度特异性,与肠道病毒EV71型,柯萨奇A、B组,麻疹病毒,风疹病毒,流感病毒以及相关腺病毒B、C组等均无交叉反应;检测灵敏度达36 copies/μl;反应体系具有很高的稳定性;整个操作过程仅需3 h～4 h。采用该方法对9份疑似VZV患者标本进行检测均为阳性,检测灵敏度明显高于病毒分离法、普通PCR法。结论:本研究建立的TaqMan荧光定量PCR方法特异、灵敏、高效,适用于VZV早期感染病例的快速确认以及暴发疫情的应急诊断中。Objective：To establish a TaqMan -based fluorescent quantitative PCR method for rapid detection of Varicella - zoster virus（VZV）. Methods ： The gene sequences of VZV downloaded from the Genbank database were aligned using the biologic software and the specific primers and probe were designed in the conserved region of the ORF62 gene （Encoding of immediate early protein IE62）for VZV. The reactive condition was optimized to improve the sensitivity and specificity of the assay. Results： Only cross reaction was observed with EV71, Cox Casset A and B VZV Oka strains generated fluorescent signals, and no group, measles virus, rubella virus, Influenza virus and adenovirus B and C group. The sensitivity was 36 copies/txl. It took only three to four hours to finish the operation. Nine clinical samples collected from VZV patients were detected for VZV by fluorescent quantitative PCR and all samples were positive. It was more sensitive than virus isolation and general PCR method. Conclusion： The TaqMan - based fluorescent quantitative PCR assay was a quick, sensitive and specific method for early clinical detection and emergent examination of VZV.

关 键 词：荧光定量PCR TAQMAN探针 水痘-带状疱疹病毒 ORF62 

分 类 号：R446.5[医药卫生—诊断学]