Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of Japanese Encephalitis Virus in Swine and Mosquitoes

Japanese Encephalitis (JE) can infect many agriculturally important animals and humans and has a high incidence in Asia. One of the natural hosts of the mosquito-borne JE Virus (JEV) is domestic pigs which act as amplifier hosts. Porcine infection results in fatal encephalitis, abortion and stillbirth in pregnant sows and hypospermia in boars. In this study, a rapid JEV Detection Method for swine and mosquitoes was developed based upon Reverse Transcription Loop-Mediated isothermal Amplification (RT-LAMP) targeting the nucleocapsid (E) genes of JEV genotype I (lineage K94PO5) and genotype III (lineage SA14-14-2). About 56 swine blood samples and 20000 mosquitoes were used to evaluate the method, compared to conventional RT-Polymerase Chain Reaction (PCR) and real-time RT-PCR. RT-LAMP had detection limits of 2.57 and 2.34 copies/mu L for JEV I and III, respectively. Assay sensitivity was similar to real-time RT-PCR but was 10 fold higher than conventional RT-PCR. Assay specificity was high, showing no cross-reactivity to other flaviviruses. Finally, the JEV RT-LAMP assay was simpler and less time consuming than conventional RT-PCR or real-time RT-PCR, since the amplification step could be completed in. a single tube within. 50 min at 63 degrees C. In conclusion, the newly-developed RT-LAMP assay is an accurate and convenient method for rapid and sensitive diagnosis of JEV in swine and mosquitoes and may prove to be a practical molecular tool for surveillance and epidemiologic investigations.