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Journal of Clinical Microbiology, June 2005, p. 2895-2903, Vol. 43, No. 6
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.6.2895-2903.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Rapid Detection and Differentiation of Dengue Virus Serotypes by a Real-Time Reverse Transcription-Loop-Mediated Isothermal Amplification Assay

Manmohan Parida,1 Kouhei Horioke,1 Hiroyuki Ishida,1 Paban Kumar Dash,2 Parag Saxena,2 Asha Mukul Jana,2 Mohammed Alimul Islam,1 Shingo Inoue,1 Norimitsu Hosaka,3 and Kouichi Morita1,4*

Department of Virology, Institute of Tropical Medicine, Nagasaki University, 1-12-4, Sakamoto, Nagasaki 852-8523, Japan,1 Department of Virology, Defense R & D Establishment, Jhansi Road, Gwalior-474002, M. P., India,2 Eiken Chemical Co. Ltd., 1381-3 Shimoishigami, Ohtawara, Tochigi 324-0036, Japan,3 CREST, Japan Science and Technology Corporation, Saitama 332-0012, Japan4

Received 15 November 2004/ Returned for modification 22 December 2004/ Accepted 2 February 2005

The development and validation of a one-step, real-time, and quantitative dengue virus serotype-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the 3' noncoding region for the rapid detection and differentiation of dengue virus serotypes are reported. The RT-LAMP assay is very simple and rapid, wherein the amplification can be obtained in 30 min under isothermal conditions at 63°C by employing a set of four serotype-specific primer mixtures through real-time monitoring in an inexpensive turbidimeter. The evaluation of the RT-LAMP assay for use for clinical diagnosis with a limited number of patient serum samples, confirmed to be infected with each serotype, revealed a higher sensitivity by picking up 100% samples as positive, whereas 87% and 81% of the samples were positive by reverse transcription-PCR and virus isolation, respectively. The sensitivity and specificity of the RT-LAMP assay for the detection of viral RNA in patient serum samples with reference to virus isolation were 100% and 93%, respectively. The optimal assay conditions with zero background and no cross-reaction with other closely related members of the Flavivirus family (Japanese encephalitis, West Nile, and St. Louis encephalitis viruses) as well as within the four serotypes of dengue virus were established. None of the serum samples from healthy individuals screened in this study showed any cross-reaction with the four dengue virus serotype-specific RT-LAMP assay primers. These findings demonstrate that RT-LAMP assay has the potential clinical application for detection and differentiation of dengue virus serotypes, especially 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, June 2005, p. 2895-2903, Vol. 43, No. 6
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.6.2895-2903.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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