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Detection of Middle East respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Kazuya Shirato1*, Takuya Yano2, Syouhei Senba3, Shigehiro Akachi2, Takashi Kobayashi2, Takamichi Nishinaka2, Tsugunori Notomi3 and Shutoku Matsuyama1

Author Affiliations

1 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

2 Mie Prefecture Health and Environment Research Institute, 3684-11 Sakura-cho, Yokkaichi, Mie 512-1211, Japan

3 Eiken Chemical Co. Ltd., 4-19-9 Taito, Taito-ku, Tokyo 110-8408, Japan

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Virology Journal 2014, 11:139  doi:10.1186/1743-422X-11-139

Published: 8 August 2014

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.

Keywords:
Meddle East respiratory syndrome (MERS); MERS coronavirus (MERS-CoV); RT-LAMP; Genetic diagnostic method