人双埃克病毒SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立及初步应用

为建立一种能快速、简便、灵敏地检测人双埃克病毒(Human Parechovirus,HPeV)SYBR Green I实时荧光定量RT-PCR检测方法,根据GenBank发表的人双埃克病毒8个基因型的参考毒株全序列,针对5′端非编码区保守序列设计1对特异性引物,通过RT-PCR扩增此靶序列,构建重组质粒作为标准阳性模板,经过优化反应条件,建立标准曲线和融解曲线,对其灵敏性、特异性和重复性进行评价;检测临床粪便样品共156份,并与常规套式RT-PCR检测结果进行比较。结果显示,建立的标准曲线Ct值与模板浓度呈现良好的线性关系(r2=0.999),融解曲线特异,检测灵敏度可达60拷贝/μL,比常规PCR检测方法高100倍,重复性变异系数小(<1%),能特异地检测HPeV。临床样本检测结果也表明该法(检出30份)灵敏度高于套式RT-PCR(检出21份)。实验结果证明建立的实时荧光定量RT-PCR检测方法具有良好的特异性、敏感性和重复性,有望成为HPeVs的分子诊断、流行病学调查等相关研究的工具。

Development and Preliminary Application of SYBR Green Ⅰ Real-Time RT-PCR Assay for Detection of HPeVs

To develop a rapid,simple and sensible SYBR Green Ⅰ real-time quantitative RT-PCR assay for detecting human parechoviruses(HPeVs),based on the genome sequences of the eight HPeVs genotypes available in GenBank,a pair of specific primers targeting the conserved 5′ untranslated region(5′UTR) were designed for amplifying 234 bp aimed DNA fragments with RT-PCR,which were then cloned into pMD18-T vector and used as standard control.By optimizing the reaction parameters,the standard curve was constructed and the melting curve was analyzed,meanwhile,the specificity,sensitivity and reproducibility were evaluated.The standard curve showed a good linear relationship between initial templates numbers and Ct values,with an excellent correlation coefficient(r2=0.999) and specific melting curve.The assay had a detection threshold of 60 copies/μL of initial templates,which was 100 times more sensitive than conventional PCR.Moreover,the variability of repetitive Ct value less than 1% exhibited a good reproducibility.156 fecal samples were detected by this established assay,and the result showed a higher sensitivity compared with routine nested RT-PCR.This assay has high sensitivity,specificity,reproducibility and simple procedure,hopefully it can be used in HPeVs clinical diagnosis and epidemical investigation.