A new molecular identification tool for biting midges of the Obsoletus group (Culicoides, Ceratopogonidae) : the ‘glass slide’ microarray approach

Abstract

Culicoides species of the Obsoletus group (Diptera: Ceratopogonidae) are potential vectors of the bluetongue virus serotype 8 (BTV 8) that was introduced into central Western Europe in 2006. A major difficulty encountered is the correct morphological species identification of the Obsoletus group females, making molecular identification the method of choice. In this study we present a new molecular tool based upon probe hybridization using a DNA microarray format, to identify Culicoides species of the Obsoletus group. The Internal Transcribed Spacer 1 (ITS1) gene sequences of 55 Culicoides belonging to 13 different species were determined and used, together with 19 Culicoides ITS1 sequences from GenBank, to design species-specific probes for the microarray test. Evaluation of this test was performed using the amplified ITS1 sequences of another 85 Culicoides specimens, belonging to 11 species. The microarray test successfully identified all samples (100%) of the Obsoletus group, specifying each specimen to species level within the group. This test has several advantages over the existing PCR based molecular tools: the possibility for parallel analysis of many species, the high sensitivity and specificity, and low background signal noise. Hand-spotting of the microarray slide and the use of detection chemistry make this alternative technique affordable and feasible for any diagnostic laboratory with PCR facilities.