Abstract:To establish a RT-heminested-PCR method for detecting flavivirus in Mosquitoes. We designed two pairs of universal primers targetting the conserved regions within nonstructural proteins gene ( NS5) of the Flavivirus genus. cDNA of JEV and DENV types I~IV were used as template to optimize the reaction conditions. The sensitivities of the assay was evaluated with attenuated vaccine strain of JEV (SA14-14 -2). Furthermore, we validated the RT-heminested-PCR assay with field-caught mosquitoes grouped in pools. The threshold concentration was 1.0×10-2 PFU/mL for detecting JEV in pool of 50 mosquitoes. RT-hemi-nested PCR assay was used to detect Culex tritaeniorhynchus collected from Puer city of YunNan province in China. Amplified sequences were identified by sequencing. Of the 54 pools tested, 9 pools contained JEV, 3 pools contained DENV type II, 1 pool contained DENV type I, and 1 pool was positive for a previously undescribed flaviviral sequence that was most similar to Quang Binh virus. These data suggest that the RT-heminested-PCR using genus universal primers can be adopted for flavivirus surveillance in mosquito populations, and this approach has the potential to detect both previously recognized and novel viruses.