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

DOI Web Site 21 Citations
  • Manmohan Parida
    Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan
  • Guillermo Posadas
    Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan
  • Shingo Inoue
    Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan
  • Futoshi Hasebe
    Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan
  • Kouichi Morita
    Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan

Abstract

<jats:title>ABSTRACT</jats:title> <jats:p> 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 <jats:italic>Bst</jats:italic> 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 <jats:sup>4</jats:sup> 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 <jats:italic>Flavivirus</jats:italic> 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. </jats:p>

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