Author
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Kato, Cecilia |
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MAYER, RICHARD - 5410-10-00 |
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Submitted to: Journal of Virological Methods
Publication Type: Other Publication Acceptance Date: 10/26/2007 Publication Date: 10/26/2007 Citation: Kato, C.Y., Mayer, R.T. An Improved, High-Throughput Method for Detection of Bluetongue Virus RNA in Culicodes Midges. Journal of Virological Methods. Presentation at the Universite Pierre and Marie Curie. Paris, France. Oct. 26, 2007. Interpretive Summary: We have developed an assay for Bluetongue virus detection using infrared-dye-labeled primers for reverse transcriptase PCR. This assay is fast, sensitive, and cost effective. We also improved the processing procedure of individual midges so that they can be processed (from homogenization to nucleic acid isolation) in 96-well plate format. Previous processing methods handled one specimen at a time. 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. |
