Shirato Kazuya, Yano Takuya, Senba Syouhei, Akachi Shigehiro, Kobayashi Takashi, Nishinaka Takamichi, Notomi Tsugunori, Matsuyama Shutoku
Laboratory of Acute Respiratory Viral Diseases and Cytokines, Department of Virology III, National Institute of Infectious Disease, Laboratory of Acute Respiratory Viral Diseases and Cytokines, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.
Virol J. 2014 Aug 8;11:139. doi: 10.1186/1743-422X-11-139.
The first documented case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) occurred in 2012, and outbreaks have continued ever since, mainly in Saudi Arabia. MERS-CoV is primarily diagnosed using a real-time RT-PCR assay, with at least two different genomic targets required for a positive diagnosis according to the case definition of The World Health Organization (WHO) as of 3 July 2013. Therefore, it is urgently necessary to develop as many specific genetic diagnostic methods as possible to allow stable diagnosis of MERS-CoV infections.
Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) is a genetic diagnostic method used widely for the detection of viral pathogens, which requires only a single temperature for amplification, and can be completed in less than 1 h. This study developed a novel RT-LAMP assay for detecting MERS-CoV using primer sets targeting a conserved nucleocapsid protein region.
The RT-LAMP assay was capable of detecting as few as 3.4 copies of MERS-CoV RNA, and was highly specific, with no cross-reaction to other respiratory viruses. Pilot experiments to detect MERS-CoV from medium containing pharyngeal swabs inoculated with pre-titrated viruses were also performed. The RT-LAMP assay exhibited sensitivity similar to that of MERS-CoV real-time RT-PCR.
These results suggest that the RT-LAMP assay described here is a useful tool for the diagnosis and epidemiologic surveillance of human MERS-CoV infections.
中东呼吸综合征冠状病毒（MERS-CoV）的首例记录病例于2012年出现，此后疫情持续不断，主要发生在沙特阿拉伯。MERS-CoV主要通过实时逆转录聚合酶链反应（RT-PCR）检测进行诊断，根据世界卫生组织（WHO）截至2013年7月3日的病例定义，阳性诊断需要至少两个不同的基因组靶点。因此，迫切需要开发尽可能多的特异性基因诊断方法，以实现对MERS-CoV感染的稳定诊断。
逆转录环介导等温扩增（RT-LAMP）是一种广泛用于检测病毒病原体的基因诊断方法，该方法扩增仅需一个温度，且可在1小时内完成。本研究开发了一种新型RT-LAMP检测方法，使用靶向保守核衣壳蛋白区域的引物组来检测MERS-CoV。
RT-LAMP检测方法能够检测低至3.4拷贝的MERS-CoV RNA，且具有高度特异性，与其他呼吸道病毒无交叉反应。还进行了从接种了预先滴定病毒的咽拭子培养基中检测MERS-CoV的初步实验。RT-LAMP检测方法的灵敏度与MERS-CoV实时RT-PCR相似。
这些结果表明，本文所述的RT-LAMP检测方法是诊断和流行病学监测人类MERS-CoV感染的有用工具。
