Journal of Virological Methods 140 (2007) 140–147 An improved, high-throughput method for detection of bluetongue virus RNA in Culicoides midges utilizing infrared-dye-labeled primers for reverse transcriptase PCR Cecilia Y. Kato ∗ , Richard T. Mayer 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 Received 22 August 2006; received in revised form 6 November 2006; accepted 8 November 2006 Available online 21 December 2006 Abstract 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. Published by Elsevier B.V. Keywords: Bluetongue virus; Infrared-dye; Reverse transcriptase PCR; Culicoides sonorensis 1. Introduction Bluetongue virus (BTV) is the prototype Orbivirus within the Reoviridae family. The double stranded, segmented RNA arbovirus is vectored by several Culicoides spp. biting midges. BTV and Culicoides vectors exist in subtropical, tropical and temperate regions throughout the world. Changes in climatic conditions have allowed the spread of exotic strains of BTV into Europe, Asia, and North America (Gibbs and Greiner, 1994; Mellor and Wittmann, 2002; Purse et al., 2005; Tabachnick et al., 1996). BTV is on the Office International des Epizooties (OIE) list of notifiable diseases because of the severe socio-economic impacts caused by BTV outbreaks. BTV vectors have been identified as the critical component in the spread of the virus and the changing global epidemiology of BTV. OIE therefore emphasizes the need for surveillance of competent Culicoides vectors in areas at high risk for expanded ∗ Corresponding author. Tel.: +1 307 766 3624; fax: +1 307 766 3500. E-mail addresses: ckato@uwyo.edu (C.Y. Kato), dmayer@uwyo.edu (R.T. Mayer). habitation based on historical, geographic and climatic factors (MacLachlan and Osburn, 2006; OIE, 2004). The recent OIE recommendations necessitate the development of effective, reli- able, high-throughput methods to survey competent Culicoides and potential vectors for all BTV serotypes. Current protocols for BTV detection in biting midges involve time consuming steps for sample (single insect) homogeniza- tion, preparation, and assay (Wieser-Schimpf et al., 1993). For example cell culture techniques for BTV identification may not rapidly detect all BTV particles within a sample. Standard RT- PCR and nested PCR assays for BTV RNA require multiple steps for reverse transcriptase reaction, and primary and/or sec- ondary PCR amplification (Akita et al., 1992; Aradaib et al., 1998, 2003; Dangler et al., 1990a; Katz et al., 1993; Wilson and Chase, 1993). Finally, real time PCR protocols do not detect all 24 serotypes (Jimenez-Clavero et al., 2006; Orru et al., 2004; Wilson et al., 2004). Initial PCR detection assays for BTV involved either standard or single amplification reverse transcriptase PCR (RT-PCR) and have detection sensitivities of 1 pg to 17 fg BTV RNA (Akita et al., 1992; Dangler et al., 1990b; McColl and Gould, 1991; Wade- 0166-0934/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.jviromet.2006.11.009