A multiplex PCR-based method derived from random amplified polymorphic DNA (RAPD) markers for the identification of species of the Anopheles minimus group in Southeast Asia

Effective control of Anopheles minimus s.l., an important malaria vector in Southeast Asia, is based on the accurate identification of species within An. minimus complex, which cannot be distinguished using morphological characters. Derived from individual random amplified polymorphic DNA markers, sequence characterized amplified regions were analysed for the design of species-specific paired-primers. Combination of these primers resulted in the development of a simple, robust multiplex PCR able to identify both species An. minimus A and C belonging to the complex, hybrids AC, and three sympatric and closely related species, An. aconitus, An. pampanai and An. varuna. Hybrids AC do not possess alleles of both parents but exhibit novel adaptive potentials resulting from recombination among parental genes leading to hybrizyme.