Development of a real-time, differential RT-PCR TaqMan assay for lyssavirus genotypes 1, 5 and 6

A number of RT-PCR methods have been reported for the detection of rabies and rabies-related viruses. Here, a single, closed tube, non-nested RT-PCR TaqMan assay to distinguish between Classical rabies virus and European bat lyssaviruses 1 and 2 in real time is described. The TaqMan assay is rapid, sensitive, specific and allows for the genotyping of unknown isolates concomitant with the RT-PCR. It can be applied quantitatively and the use of an internal control enables the quality of the isolated template to be assessed. The efficiency and dynamic range of the PCR has been established using isolated viral RNA and cloned control template. Comparative performance of the TaqMan assay against other diagnostic methodologies for the detection of rabies virus has been determined and the assay validated against a panel of archival samples and virus of unknown genotype from both Germany and the Sudan. Despite sequence heterogeneity between the different genotypes in the N-gene, a universal forward and reverse primer set have been designed allowing simplification of previously described assays. Rapid genotyping of two recent EBLV2 cases in the U.K. in Daubenton's bats and a recently imported human case of rabies has been performed using this test.