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

Author:

Parida Manmohan1,Posadas Guillermo1,Inoue Shingo1,Hasebe Futoshi1,Morita Kouichi1

Affiliation:

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

Abstract

ABSTRACT 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, 10 4 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.

Publisher

American Society for Microbiology

Subject

Microbiology (medical)

Reference25 articles.

1. Anderson, J. F., T. G. Andreadis, C. R. Vossbrinck, S. Tirrell, E. M. Wakem, R. A. French, A. E. Garmendia, and H. J. Van Kruiningen. 1999. Isolation of West Nile virus from mosquitoes, crows, and a Cooper's hawk in Connecticut. Science286:2331-2333.

2. Beaty B. J. C. H. Calisher and R. S. Shope. 1989. Arboviruses p. 797-856. In N. J. Schmidt and R. W. Emmons (ed.) Diagnostic procedures for viral rickettsial and chlamydial infections. American Public Health Association Washington D.C.

3. Centers for Disease Control and Prevention. 2000. Update: West Nile virus activity, eastern United States, 2000. Morb. Mortal. Wkly. Rep.49:1044-1047.

4. Chan, A. B., and J. D. Fox. 1999. NASBA and other transcription-based amplification methods for research and diagnostic microbiology. Rev. Med. Microbiol.10:185-196.

5. Nucleic acid sequence-based amplification

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