Molecular tools for early detection of invasion of malaria vector Anopheles stephensi

There are increasing reports of the expansion of the Asian malaria vector Anopheles stephensi in new geographical areas posing a threat to the elimination of urban malaria. Efficient surveillance of this vector in affected areas and early detection in new geographical areas is key for the containment and control of this species. To overcome the practical difficulties associated with the morphological identification of immature stages and adults of An. stephensi, a species-specific PCR and a Real-Time PCR assay were developed for their identification targeting a unique segment of ITS2 lacking homology to any other organism. Both PCRs can be used to identify An. stephensi individually or in pooled samples of mixed species, including when present in extremely low proportion. This study also reports a method for selective amplification and sequencing of An. stephensi for their confirmation in pooled samples of mixed species.