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Journal of Clinical Microbiology, January 2004, p. 257-263, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.257-263.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of West Nile Virus

Manmohan Parida, Guillermo Posadas, Shingo Inoue, Futoshi Hasebe, and Kouichi Morita*

Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan

Received 14 July 2003/ Returned for modification 4 October 2003/ Accepted 13 October 2003

A one-step, single tube, real-time accelerated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the envelope gene of West Nile (WN) virus. The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, efficiency, and rapidity under isothermal conditions with a set of six specially designed primers that recognize eight distinct sequences of the target. The whole procedure is very simple and rapid, and amplification can be obtained in less than 1 h by incubating all of the reagents in a single tube with reverse transcriptase and Bst DNA polymerase at 63°C. Detection of gene amplification could be accomplished by agarose gel electrophoresis, as well as by real-time monitoring in an inexpensive turbidimeter. When the sensitivity of the RT-LAMP assay was compared to that of conventional RT-PCR, it was found that the RT-LAMP assay demonstrated 10-fold higher sensitivity compared to RT-PCR, with a detection limit of 0.1 PFU of virus. By using real-time monitoring, 104 PFU of virus could be detected in as little as 17 min. The specificity of the RT-LAMP assay was validated by the absence of any cross-reaction with other, closely related, members of the Flavivirus group, followed by restriction digestion and nucleotide sequencing of the amplified product. These results indicate that the RT-LAMP assay is extremely rapid, cost-effective, highly sensitive, and specific and has potential usefulness for rapid, comprehensive WN virus surveillance along with virus isolation and/or serology.

* 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, January 2004, p. 257-263, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.257-263.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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