Development of DNA barcode-based PCR methodology to distinguish two sympatric species viz. Phlebotomus argentipes and Phlebotomus colabaensis (Diptera: Psychodidae: Phlebotominae)

Phlebotomus argentipes and Phlebotomus colabaensis are the two sandfly species prevalent in the Western Ghats region of Kerala. P. argentipes had been incriminated as the vector of leishmaniasis in this region. The similar taxonomic features shared by these two species of sandflies had made it difficult to distinguish the adults morphologically. DNA barcoding is an efficient molecular diagnostic method utilized to identify the closely related species. In the present study, we designed species specific DNA primers from the DNA barcode sequences of P. argentipes and P. colabaensis . A semi nested PCR was performed with a consensus forward primer and two different reverse primers which could amplify specific DNA fragments for these two different sandflies. An amplified PCR product of 465 bp indicated P. argentipes and 401 bp, P. colabaensis . This species-specific PCR assay can be utilized to identify and distinguish two common Phlebotomus species with a single-step multiplex PCR.