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.
Kato
Cecilia Y
CY
U.S. Department of Agriculture, Agricultural Research Service, Arthropod-Borne Animal Diseases Research Laboratory, College of Agriculture D3354, 1000 East University Avenue, Laramie, WY 82072, USA. ckato@uwyo.edu
Mayer
Richard T
RT
D000818:Animals; D001819:Bluetongue / Q000175:diagnosis / Q000821:virology*; D001820:Bluetongue virus / Q000145:classification / Q000235:genetics*; D002519:Ceratopogonidae / Q000821:virology*; D017931:DNA Primers / Q000737:chemistry*; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping; D013997:Time Factors
D017931:DNA Primers; D012367:RNA, Viral