title: Development of a real-time, TaqMan reverse transcription-PCR assay for detection and differentiation of lyssavirus genotypes 1, 5, and 6.
abstract: 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.
authors.affiliation: World Health Organization Collaborating Centre for the Characterization of Rabies and Rabies-Related Viruses, Department of Virology, Veterinary Laboratories Agency, Addlestone, United Kingdom. p.wakeley@vla.defra.gsi.gov.uk
authors.forename: P R
authors.initials: PR
authors.lastname: Wakeley
authors.forename: N
authors.initials: N
authors.lastname: Johnson
authors.forename: L M
authors.initials: LM
authors.lastname: McElhinney
authors.forename: D
authors.initials: D
authors.lastname: Marston
authors.forename: J
authors.initials: J
authors.lastname: Sawyer
authors.forename: A R
authors.initials: AR
authors.lastname: Fooks
mesh_terms: D000818:Animals; D002415:Cats; D002417:Cattle; D002460:Cell Line; D002685:Chiroptera / Q000821:virology; D004285:Dogs; D005060:Europe; D005838:Genotype; D006801:Humans; D018114:Lyssavirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D011818:Rabies / Q000821:virology; D011820:Rabies virus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D018353:Rhabdoviridae Infections / Q000821:virology; D019914:Taq Polymerase / Q000378:metabolism*
chemical_list: D012367:RNA, Viral; D019914:Taq Polymerase