TaqMan-MGB荧光定量聚合酶链反应检测水痘-带状疱疹病毒

目的利用TaqMan-MGB探针技术,建立水痘-带状疱疹病毒(VZV)的荧光定量聚合酶链反应(PCR)检测方法,用于临床标本VZV的检测。方法从GenBank上下载几十株VZV基因组序列,在IE62基因的保守区域设计引物与TaqMan-MGB探针,并对反应条件和体系进行优化,同时评价建立检测体系的特异性、敏感性和重复性;利用已知拷贝数的质粒标准品的病毒脱氧核糖核酸(DNA),建立了VZV基因拷贝数定量分析模型。结果该方法与引起相似发病症状的肠道病毒71型,柯萨奇病毒A、B组以及腺病毒均无交叉反应,具有很高的特异性,且灵敏度达到102拷贝,优于常规PCR方法。采用该法对8份疑似水痘、带状疱疹患者疱疹液标本检测均为阳性,较病毒分离、常规PCR法更为简便、快速、敏感。结论该方法适合临床早期感染病例的确认以及水痘、带状疱疹爆发疫情的应急诊断。

Detection of Varicella-zoster Virus by TaqMan-MGB Real-time PCR

Objective To develop a real-time PCR method using TaqMan-MGB probe to detect Varicella-zoster virus(VZV) in clinical specimens. Methods The VZV real-time PCR primer and probe were designed from the conservative region of IE 62 gene sequences of VZV genome downloaded from GenBank database.The real-time PCR reactive system and thermal cycle temperature were optimized and specificity,sensitivity and repeatability of the method were evaluated.The VZV quantitative analysis model were constructed using ten-fold serial dilutions of the standard plasmids. Results This method was very specific and had no cross reaction with intestinal virus EV71,Cox Casset A and B group and adenovirus.The assay was able to detect 100 copies of VZV plasmids and was better than conventional PCR method.Eight clinical samples collected from Varicella-zoster patients were detected for VZV by real-time PCR and all samples were positive.It was quicker and more sensitive than virus isolation and conventional PCR method. Conclusion This TamMan-MGB method provides rapid,specific, and sensitive alternative for VZV detectim in clinical specimens and VZV epidemic.