Development of Quantitative Real-Time PCR Tests for the Identification of Biting Midge Species and Clades (Diptera: Ceratopogonidae) of the Obsoletus Group (Subgenus Avaritia), Including Important Viral Vectors in Europe

Species of the widespread Obsoletus Complex (<i>Culicoides</i> subgenus <i>Avarita</i> Fox, 1955) have been implicated as potential key vectors during the bluetongue and Schmallenberg epidemics in Central Europe in 2006 and 2012. Although extensive efforts have been made to clarify vector-pathogen relationships, one of the most important steps in this process-correct species identification-remains difficult, due to the presence of isomorphic species within the Obsoletus Group. To overcome the difficulties in morphological species identification, several PCR tests were developed. With the aim of developing a high-throughput PCR, capable of differentiating all putative vector species and newly described haplotypes of the subgenus <i>Avaritia</i> present in Europe, a dataset of 4407 published sequences of the mitochondrial (mt) cytochrome c oxidase subunit I (COI) was used to develop specific primers and probes, which can either be applied in a singleplex PCR or in different multiplex PCR approaches. The real-time PCR achieved very high diagnostic sensitivity (100%) and specificity (91.7%) and reliably detected the three clades of <i>C. obsoletus</i> sensu stricto (s.s.) in a pool of specimens. Thus, the new real-time PCR approach will provide an excellent tool for large-scale monitoring, which could improve the understanding of the biology, geographical distribution, and habitat preference of European biting midge species involved in the transmission of bluetongue, Schmallenberg, and epizootic hemorrhagic disease viruses.