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

Authors: Kato, Cecilia; Mayer, Richard

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/8/2006
Publication Date: 3/1/2007
Citation: Kato, C.Y., Mayer, R.T. 2007. An improved, high-throughput method for detection of bluetongue virus RNA in Culicoides midges utilizing infrared-dye-labeled primers for reverse transcriptase PCR. J. of Virological Methods, V. 140, Issues 1-2, March 2007, p. 140-147.

Interpretive Summary: A fast and precise assay, specific to all 24 serotypes of bluetongue virus (BTV) is essential for accurate screening and survillance for this economically important world-wide arbovirus. A new rapid (less than 6 h start-to-finish) high-throughput assay is reported that is sensitive and specific for detecting BTV RNA in Culicoides biting midges. 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 rapid RNA extraction. The RNA was used in a single step reverse transcriptase PCR (RT-PCR) reaction with infrared (IR)-dye-labled primers. The RT-PCR products were visualized in agarose gels by an infrared scanner. The adaptation of IR-dye-labeled primers and 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.

Technical Abstract: A new rapid (less than 6 h from insect-to-results) high-throughput assay is reported that is sensitive and specific for detecting BTV RNA in Culicoides biting midges. 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.