[Application of one step RT-PCR assay for detection of flavivirus RNA in mosquitoes].

The conditions of one step RT-PCR method for detection of virus RNA in field-collected mosquitoes, and preservation period of infected mosquitoes for one step RT-PCR were examined. We compared several virus RNA extraction methods with artificially contaminated mosquito pools with dengue virus (DV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV) with a known amount of plaque forming unit (PFU) to establish the condition of one step RT-PCR. In this study, most effective RNA extraction method was ISOGEN-LS extraction combined with supernatant of centrifuged mosquito homogenates. Detection limit of one step RT-PCR using flavivirus universal primer in ten mosquitoes/tube (pool) was 10 PFU of DV, JEV and YFV, 1 PFU of each viruses using species-specific primer respectively, in one hundred mosquitoes/tube, 100 PFU/tube using universal primer pairs, 10 PFU/tube using species-specific primer pairs respectively. Dengue virus infected single mosquito was mixed with 99 un-infected mosquitoes, and tested by one step RT-PCR. We could detect single infected mosquito in pools containing 99 un-infected mosquitoes. Aedes aegypti and Aedes albopictus were inoculated intrathoracically with a mouse-adapted strain of dengue-1 virus and were kept up to 30 days at different temperature. Then examined by one step RT-PCR to determine the appropriate mosquito handling method and the condition of transportation. Positive result was obtained up to 30 days after the mosquito died naturally. These results suggested that we could detect flavivirus RNA tested not only from live mosquitoes but also dead mosquitoes as well, and could apply one step RT-PCR as a rapid, specific, and highly sensitive tool for flavivirus surveillance.