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Journal of Clinical Microbiology, November 2006, p. 4172-4178, Vol. 44, No. 11
0095-1137/06/$08.00+0     doi:10.1128/JCM.01487-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid and Real-Time Detection of Japanese Encephalitis Virus

Division of Virology, Defence Research & Development Establishment, Gwalior, Madhya Pradesh 474002, India,¹ Department of Virology, Institute of Tropical Medicine, Nagasaki University, 1-12-4, Sakamoto, Nagasaki, 852-8523, Japan²

Received 19 July 2006/ Returned for modification 27 August 2006/ Accepted 4 September 2006

The standardization and validation of a one-step, single-tube accelerated quantitative reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay is reported for rapid and real-time detection of Japanese encephalitis virus (JEV). The RT-LAMP assay reported in this study is very simple and rapid; the amplification can be obtained in 30 min under isothermal conditions at 63°C by employing a set of six primers targeting the E gene of JEV. The RT-LAMP assay demonstrated exceptionally higher sensitivity compared to that of RT-PCR, with a detection limit of 0.1 PFU. The specificities of the selected primer sets were established by cross-reactivity studies with other closely related members of the JEV serocomplex as well as by evaluation of healthy human volunteers. The comparative evaluation of the RT-LAMP assay for clinical diagnosis with a limited number of patient cerebrospinal fluid samples revealed 85% concordance with conventional RT-PCR, with a sensitivity and a specificity of 100% and 86%, respectively. The concentration of virus in most of the clinical samples was 10² to 10⁵ PFU/ml, as determined from the standard curve based on the time of positivity in the samples. In addition, the monitoring of gene amplification can also be visualized with the naked eye by using SYBR green I fluorescent dye. Thus, due to easy operation without a requirement of sophisticated equipment and skilled personnel, the RT-LAMP assay reported here is a valuable tool for the rapid and real-time detection of JEV not only by well-equipped laboratories but also by peripheral diagnostic laboratories with limited financial resources in developing countries.

Published ahead of print on 27 September 2006.