PCR-based methods for identification of species of the Anopheles minimus group: allele-specific amplification and single-strand conformation polymorphism

We report two polymerase chain reaction (PCR)-based methods for distinguish ing morphologically similar species based on amplification of a variable re gion of the 28S gene of ribosomal DNA. The four species we investigated are mosquitoes of the Anopheles minimus group: An, aconitus, An. varuna and An . minimus species A and C. The formally named species are vectors of human malaria parasites in south-east Asia but are difficult to distinguish with certainty on the basis of morphology. Allele-specific amplification was use d to differentiate An. minimus A from An. minimus C. This technique has bee n widely used for the diagnosis of species. Single-strand conformation poly morphisms (SSCPs) were used to separate all four species. This technique, w hich has seldom been used for species identification, has many advantages: it does not require sequence information beyond that needed for amplificati on; it is ideally suited for the detection of heterozygotes; it utilizes mo re of the information in the PCR product than allele-specific amplification ; it distinguishes all four species considered here and could easily be ext ended to other species; previously unknown intraspecific variation and addi tional species are likely to be detected. Thus, SSCPs provide valuable popu lation genetic information which allele-specific amplification does not.