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

• Published in: Journal of Clinical Microbiology Vol. 43; no. 6; pp. 2786 - 2792
• Main Authors: Wakeley, P. R, Johnson, N, McElhinney, L. M, Marston, D, Sawyer, J, Fooks, A. R
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.06.2005
• Subjects: Animals; Biological and medical sciences; Cats; Cattle; Cell Line; Chiroptera - virology; Dogs; Europe; Fundamental and applied biological sciences. Psychology; Genotype; Humans; Infectious diseases; Lyssavirus; Lyssavirus - classification; Lyssavirus - genetics; Lyssavirus - isolation & purification; Medical sciences; Mice; Microbiology; Miscellaneous; Rabies - virology; Rabies virus; Rabies virus - classification; Rabies virus - genetics; Rabies virus - isolation & purification; Reverse Transcriptase Polymerase Chain Reaction - methods; Rhabdoviridae Infections - virology; RNA, Viral - analysis; RNA, Viral - isolation & purification; Taq Polymerase - metabolism; Virology; Europe; Virus; Mononegavirales; Microbiology; Rhabdoviridae; Genotype; Lyssavirus; Detection; Real time; Reverse transcription polymerase chain reaction
• ISSN: 0095-1137; 1098-660X
• DOI: 10.1128/JCM.43.6.2786-2792.2005

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.