Rapid differential detection of genotype I and III Japanese encephalitis virus from clinical samples by a novel duplex TaqMan probe-based RT-qPCR assay

•Rapid differention of Genotype I and III Japanese Encephalitis Virus by a novel duplex TaqMan probe-based RT-qPCR assay.•Rapid detection of Japanese Encephalitis Virus in clinical samples from pigs and mosquitoes.•Co-circulation of GI and GIII infections with GI infection being more prevalent in pigs or mosquitoes in eastern China. Japanese Encephalitis (JE) is an acute infectious disease that threatens both human and pig populations throughout Asia. JE is caused by the Japanese Encephalitis Virus (JEV), of which genotype III (GIII) had been the most prevalent strain throughout Asia, but recent studies have shown that genotype I (GI) has replaced GIII as the predominant version. Pigs and mosquitoes play a primary role in JEV transmission. However, a method for the rapid differentiation between JEV G I and G III remains unavailable. This study aimed to establish a rapid JEV genotyping method using novel duplex TaqMan RT-qPCR assay.specific primer and probes located in the PrM/M gene that were able to specifically differentiate GI and GIII JEV, was selected as the duplex TaqMan RT-qPCR target.The specificity, sensitivity and reproducibility test of this assay were validated. The sensitivity of the assay was 10 genomic RNA copies for both GI and GIII JEV in field mosquito and pig samples,and more sensitive than the current methods. In addition, the novel assay can be completed in less than 1 h. Therefore, This duplex TaqMan RT-qPCR assay is a promising tool for rapid differential detection and epidemiology of GI and GIII JEV strains in China. The results showed that co-circulation of GI and GIII infections with GI infection being more prevalent in pigs or mosquitoes in eastern China.