title: Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus.
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 degrees 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.
authors.affiliation: Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan.
authors.forename: Manmohan
authors.initials: M
authors.lastname: Parida
authors.forename: Guillermo
authors.initials: G
authors.lastname: Posadas
authors.forename: Shingo
authors.initials: S
authors.lastname: Inoue
authors.forename: Futoshi
authors.initials: F
authors.lastname: Hasebe
authors.forename: Kouichi
authors.initials: K
authors.lastname: Morita
mesh_terms: D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014902:West Nile virus / Q000235:genetics / Q000302:isolation & purification*