Rapid detection and quantification of Japanese encephalitis virus by real-time reverse transcription loop-mediated isothermal amplification.

We established a rapid, quantitative real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the envelope gene of Japanese encephalitis virus. The RT-LAMP enabled us to detect the target product within 1 hr by only reacting reverse transcriptase and Bst DNA polymerase in a single tube at an isothermal temperature. The detection sensitivity of the RT-LAMP for Japanese encephalitis virus was 1 PFU, similar to that of conventional reverse transcription-polymerase chain reaction (RT-PCR). Flaviviruses of the Japanese encephalitis virus group, such as Dengue virus and West Nile virus, could not be detected. This confirmed the specificity of the RT-LAMP assay for Japanese encephalitis virus. A standard curve was constructed by plotting viral titer against the time for virus detection by the RT-LAMP, validating the quantitative accuracy of the assay. In addition, the amount of virus estimated by RT-LAMP was strongly correlated (r = 0.902) with that determined by plaque assay, a conventional method for virus quantification. These results indicate that the RT-LAMP assay established in this study is specific for Japanese encephalitis virus, and allows more rapid detection and quantification of the virus.