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Journal of Clinical Microbiology, May 2004, p. 1956-1961, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.1956-1961.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Method for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus

Hong Thi Cam Thai,1 Mai Quynh Le,2 Cuong Duc Vuong,2 Manmohan Parida,1 Harumi Minekawa,3 Tsugunori Notomi,3 Futoshi Hasebe,1 and Kouichi Morita1*

Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523,1 Eiken Chemical Co. Ltd., Ohtawara, Tochigi 324-0036, Japan,3 Department of Virology, National Institute of Hygiene and Epidemiology, Hanoi, Vietnam2

Received 7 November 2003/ Returned for modification 27 January 2003/ Accepted 2 February 2004

The development and evaluation of a one-step single-tube accelerated real-time quantitative reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay is reported for rapid detection of the severe acute respiratory syndrome coronavirus (SARS-CoV) replicase gene. A total of 49 samples (15 throat washes, 13 throat swabs, and 21 combined throat and nasal swabs) collected from patients admitted to the Hanoi-French and Ninhbinh hospitals in Vietnam during the SARS epidemic were evaluated and compared to conventional RT-PCR. The RT-LAMP assay demonstrated 100-fold-greater sensitivity, with a detection limit of 0.01 PFU. The sensitivity and specificity of RT-LAMP assay for detecting viral RNA in clinical specimens with regard to RT-PCR were 100 and 87%, respectively. The specificity of the RT-LAMP assay was further validated by restriction analysis as well as nucleotide sequencing of the amplified product. The concentration of virus in most of the clinical samples was 0.1 PFU (0.1 to 102 PFU), as determined from the standard curve of SARS RT-LAMP and based on the time of positivity. The assay procedure is quite simple, wherein the amplification is carried out in a single tube under isothermal conditions at 63°C, and the result can be obtained in less than 1 h (as early as 11 min). Thus, the RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection and will be useful for rapid and reliable clinical diagnosis of SARS-CoV in developing countries.

* Corresponding author. Mailing address: Department of Virology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. Phone: 81 95 849 7829. Fax: 81 95 849 7830. E-mail: moritak{at}net.nagasaki-u.ac.jp.

Journal of Clinical Microbiology, May 2004, p. 1956-1961, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.1956-1961.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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