An improved, high-throughput method for detection of bluetongue virus RNA in Culicoides midges utilizing infrared-dye-labeled primers for reverse transcriptase PCR.

A new rapid (less than 6h from insect-to-results) high-throughput assay that is sensitive and specific for detecting BTV RNA in Culicoides biting midges is reported. Homogenization and extraction of nucleic acids from individual Culicoides specimens were performed in a 96-well plate format using specialized beads in a homogenization buffer compatible with cell culture and RNA extraction. A portion of homogenate (10%) from each specimen was retained for confirmatory infectious virus isolation, while the remaining 90% was used for RNA extraction. The RNA was used in a single step reverse transcriptase PCR (RT-PCR) reaction with infrared (IR)-dye-labeled primers. The RT-PCR products were visualized in agarose gels with an infrared scanner. The adaptation of IR-dye-labeled primers in combination with a one step RT-PCR resulted in a detection limit of 0.5 pfu of purified BTV RNA. All 24 serotypes of BTV prototype strains and none of the 8 serotypes of the closely related epizootic hemorrhagic disease virus (EHDV) prototype strains were detected.