Analytical & Measuring Instruments Division, Shimadzu Corporation, 1 Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto 604-8511, Japan.
J Virol Methods. 2010 Feb;163(2):282-6. doi: 10.1016/j.jviromet.2009.10.011. Epub 2009 Oct 28.
Noroviruses are important human pathogens which cause epidemic acute viral gastroenteritis. Current techniques used for detection of noroviruses in fecal specimens involve multi-step viral RNA extraction and purification followed by reverse transcriptase-polymerase chain reaction (RT-PCR). This study demonstrates a method for easy detection of norovirus in fecal specimens, involving one-step RNA release and direct use of the released RNA for RT-PCR (direct RT-PCR). For one-step RNA release, a simple method was adopted based on addition of the sample treatment reagent from a commercialized Norovirus GI and GII RNA Detection Kit to suspended fecal specimens, followed by a brief heat treatment. The released RNA was then added directly to the RT mixture from the same kit. After reverse transcription and PCR, the product was detected by agarose gel electrophoresis. Direct RT-PCR was evaluated with 275 fecal specimens comprising 230 norovirus-positive and 45 norovirus-negative samples as assessed by real-time RT-PCR, considered to be the "gold standard" for norovirus detection. Direct RT-PCR was sufficiently specific and sensitive for norovirus detection, and eliminated the RNA extraction and purification step. Use of this method should facilitate detection of norovirus in fecal specimens and provide valuable information regarding the incidence of the virus. In addition, this method should be applicable for other RNA viruses.
诺如病毒是重要的人类病原体，可引起流行的急性病毒性胃肠炎。目前用于粪便标本中检测诺如病毒的技术涉及多步病毒 RNA 提取和纯化，然后进行逆转录-聚合酶链反应（RT-PCR）。本研究展示了一种简便的粪便标本中诺如病毒检测方法，涉及一步 RNA 释放，以及直接将释放的 RNA 用于 RT-PCR（直接 RT-PCR）。对于一步 RNA 释放，采用了一种简单的方法，即在悬浮粪便标本中加入商业化的诺如病毒 GI 和 GII RNA 检测试剂盒的样品处理试剂，然后进行短暂的热处理。然后将释放的 RNA 直接加入同一试剂盒的 RT 混合物中。逆转录和 PCR 后，通过琼脂糖凝胶电泳检测产物。直接 RT-PCR 用 275 份粪便标本进行评估，其中 230 份为实时 RT-PCR 评估的诺如病毒阳性样本，45 份为诺如病毒阴性样本，被认为是诺如病毒检测的“金标准”。直接 RT-PCR 对诺如病毒的检测具有足够的特异性和敏感性，并消除了 RNA 提取和纯化步骤。该方法的使用应有助于粪便标本中诺如病毒的检测，并提供有关病毒发病率的有价值信息。此外，该方法应该适用于其他 RNA 病毒。
