Integration of Anopheles beklemishevi (Diptera: Culicidae) in a PCR assay diagnostic for palaearctic Anopheles maculipennis sibling species

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Abstract
A few years ago a PCR-based assay for a quick and reliable identification of six palaearctic sibling species of the Anopheles maculipennis complex was presented making use of differences in the nucleotide sequence of the ITS2 ribosomal mosquito DNA. An. beklemishevi, which is distributed in Scandinavia and Russia only, has now been integrated into this test after analysis of its ITS2 region which turned out to be much longer than those of the other sibling species. Three oligonucleotides putatively specific for An. beklemishevi were constructed and tested in combination with a universal genus-specific primer for the amplification of an An. beklemishevi-specific ITS2 DNA-fragment. Two of the three oligos generated accurate and specific PCR products, even when used in a multiplex PCR together with the specific primers for the other six sibling species. Cross-hybridization of the primers to heterologous culicid DNA was never observed. The amplicons that identify An. beklemishevi consist of 554 and 735 bp, respectively, and are easily distinguished from those specific for the other sibling species after gel electrophoresis.

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