检测戊型肝炎病毒核酸的实时荧光聚合酶链反应法的建立Development of Real-time Fluorescent RT-PCR for Determination of Hepatitis E Virus RNA
赵晨燕;阎宝山;张峰;李卓;张春涛;王佑春;
摘要(Abstract)：
目的建立一种能快速、敏感地检测戊型肝炎病毒(HEV)核酸的实时荧光聚合酶链反应法(Real-timeRT-PCR)。方法基于HEVORF3保守区序列,设计引物和探针,从22对引物和4条探针中进行筛选,并对选出的引物和探针的浓度、反应体系、反应条件、核酸抽提方法等进行优化。采用常规套式RT-PCR和本研究建立的实时荧光RT-PCR检测不同样品,比较两种方法的特异性和敏感性。结果优化出1对引物和探针的组合。优化的30μl反应体系中,上、下游引物浓度均为0.10mmol/L,探针浓度为0.20mmol/L,Taq酶的浓度为3U,AMV逆转录酶浓度为2U。优化的反应条件为:50℃30min;95℃3min;95℃10s,50℃30s,72℃60s,5个循环;95℃10s,57℃40s,40个循环。初步检测结果显示,该方法与常规套式RT-PCR特异性符合率为100%,两种方法检测HEV1型样品的敏感性均为104,检测人和猪的HEV4型样品时,实时荧光RT-PCR敏感性均为104,明显高于常规套式RT-PCR的103和102。结论所建立的检测HEV核酸的实时荧光聚合酶链反应法具有简便、快速、敏感性较高以及不易被污染等优点,具有一定的临床应用潜力。
关键词(KeyWords)： 戊型肝炎病毒;核糖核酸;实时荧光逆转录聚合酶链反应;优化
基金项目(Foundation): 国家863课题基金资助(2006AA02Z453).
作者(Authors): 赵晨燕;阎宝山;张峰;李卓;张春涛;王佑春;
DOI: 10.13200/j.cjb.2007.12.65.zhaochy.016
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