A Multiplex PCR Assay for Six Aedini Species, Including Aedes albopictus

Background: Mosquitoes, as vectors of various human pathogens, are significant drivers of serious human illness. In particular, those species in the Aedini tribe, which typically transmit dengue virus, Chikungunya fever virus, and Zika virus, are increasing their range because of climate change and international commerce. In order to prevent mosquito-borne disease, accurate mosquito species identification and monitoring are needed. The goal of this work was to develop a rapid and simple molecular diagnostic method for six morphologically similar Aedini species ( Aedes flavopictus , Aedes albopictus , Ochlerotatus koreicus , Ochlerotatus japonicus , Ochlerotatus togoi , and Ochlerotatus hatorii ) in Korea. Methods: In total, 109 samples were used in this study. The internal transcribed spacer 2 (ITS2) regions from all six species were amplified, sequenced and analyzed using Mega 6. Once regions that were consistently different in sequence between all six species were identified, multiplex primers were designed to amplify them to generate species-specific fragments distinguishable by their size. Results: Uniquely sized fragments were generated in Aedes flavopictus (495bp), Aedes albopictus (438bp), Ochlerotatus koreicus (361bp), Ochlerotatus togoi (283bp), Ochlerotatus hatorii (220bp), and Ochlerotatus japonicus (160bp). Pairwise distance analysis showed that the difference was 35.0±1.5% between Aedes spp. and Ochlerotatus spp., 17.4±0.2% between Aedes albopictus and Aedes flavopictus , and 11.1±0.3% between Ochlerotatus koreicus and Ochlerotatus japonicus . Conclusions: In this study, a multiplex PCR assay for six species of the Aedini tribe was developed. This assay is more accurate than morphological identification and will be useful for monitoring and controlling these vector mosquitoes.