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Journal of Clinical Microbiology, June 2005, p. 2786-2792, Vol. 43, No. 6
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.6.2786-2792.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Development of a Real-Time, TaqMan Reverse Transcription-PCR Assay for Detection and Differentiation of Lyssavirus Genotypes 1, 5, and 6

Rabies Research and Diagnostic Group, World Health Organization Collaborating Centre for the Characterization of Rabies and Rabies-Related Viruses, Department of Virology, and Technology Transfer Unit, Biotechnology Department, Veterinary Laboratories Agency, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom

Received 21 July 2004/ Returned for modification 23 November 2004/ Accepted 17 January 2005

Several reverse transcription-PCR (RT-PCR) methods have been reported for the detection of rabies and rabies-related viruses. These methods invariably involve multiple transfers of nucleic acids between different tubes, with the risk of contamination leading to the production of false-positive results. Here we describe a single, closed-tube, nonnested RT-PCR with TaqMan technology that distinguishes between classical rabies virus (genotype 1) and European bat lyssaviruses 1 and 2 (genotypes 5 and 6) in real time. The TaqMan assay is rapid, sensitive, and specific and allows for the genotyping of unknown isolates concomitant with the RT-PCR. The assay can be applied quantitatively and the use of an internal control enables the quality of the isolated template to be assessed. Despite sequence heterogeneity in the N gene between the different genotypes, a universal forward and reverse primer set has been designed, allowing for the simplification of previously described assays. We propose that within a geographically constrained area, this assay will be a useful tool for the detection and differentiation of members of the Lyssavirus genus.

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