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

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Abstract
Summary: We report two polymerase chain reaction (PCR)‐based methods for distinguishing morphologically similar species based on amplification of a variable region 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 used to differentiate An. minimus A from An. minimus C. This technique has been widely used for the diagnosis of species. Single‐strand conformation polymorphisms (SSCPs) were used to separate all four species. This technique, which has seldom been used for species identification, has many advantages: it does not require sequence information beyond that needed for amplification; it is ideally suited for the detection of heterozygotes; it utilizes more of the information in the PCR product than allele‐specific amplification; it distinguishes all four species considered here and could easily be extended to other species; previously unknown intraspecific variation and additional species are likely to be detected. Thus, SSCPs provide valuable population genetic information which allele‐specific amplification does not.