The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used. 1. Introduction Rabies diagnosis is based on fluorescent antibody testing (FAT) in brain smears and inoculation of brain suspension either in mouse neuroblastoma cell cultures or intracerebrally in mice as confirmatory assays with high sensitivity and specificity for postmortem diagnosis [1–3]. These techniques are well suited for rapid and reliable routine diagnosis, if brain material is available. For other diagnostic specimens sampled intra vitam in case of clinical suspicion, for example, saliva, cerebrospinal fluid, or skin biopsies, more suited molecular techniques with excellent sensitivity have been developed and validated, mostly targeting the conserved nucleoprotein gene of the lyssavirus genome, for example, [4–10]. Among these, there are protocols both for classical RT-PCR and for real-time RT-PCR, which adds speed, efficiency, contamination safety, and reliability to the technique combined with the potential to quantify the viral load [11, 12]. A major advantage offered by these molecular techniques is the characterisation of viral isolates by sequencing the given amplification products followed by phylogenetic or phylogeographic analysis [13–15]. The greatest
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