Beuret Christian
Spiez Laboratory, Christian Beuret, 3700 Spiez, Switzerland.
J Virol Methods. 2004 Jan;115(1):1-8. doi: 10.1016/j.jviromet.2003.09.005.
A multiplex real-time RT-PCR protocol for the simultaneous detection of noroviruses ("Norwalk-like viruses") of genogroups I and II, human astroviruses and enteroviruses is described. The protocol was developed and evaluated using the LightCycler and corresponding SYBR Green reagents. New primers were designed within conserved genome regions to optimize the detection range of virus subtypes of each genus. To enable the development of a multiplex PCR assay within one tube (capillary), similar mastermix- and cycling-conditions were respected for each individual primer system. Subsequent melting curve analysis allowed the determination of possible dual-contaminations of entero- and noro- or astroviruses by the formation of dual peaks. Special care was taken to minimize the loss of sensitivity, since the detection of small viral contaminations is a crucial parameter especially for food analysis. The multiplex assay was compared successfully to the single SYBR Green assay, and revealed to be at least 10 times more sensitive than the one obtained with an endpoint PCR thermocycler protocol published previously.
本文描述了一种用于同时检测I群和II群诺如病毒（“诺沃克样病毒”）、人星状病毒和肠道病毒的多重实时逆转录聚合酶链反应（RT-PCR）方法。该方法是使用LightCycler和相应的SYBR Green试剂开发并评估的。在保守基因组区域内设计了新引物，以优化每个属病毒亚型的检测范围。为了在一管（毛细管）内开发多重PCR检测方法，每个单独的引物系统都遵循相似的预混液和循环条件。随后的熔解曲线分析通过双峰的形成确定肠道病毒与诺如病毒或星状病毒可能的双重污染。特别注意尽量减少灵敏度的损失，因为检测少量病毒污染是一个关键参数，尤其是对于食品分析而言。该多重检测方法与单SYBR Green检测方法成功进行了比较，结果显示其灵敏度至少比之前发表的终点PCR热循环仪方法高10倍。
