A novel multiplex RT ‐ qPCR method based on dual‐labelled probes suitable for typing all known genotypes of viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia ( VHS ) is a notifiable fish disease, whose causative agent is a rhabdovirus isolated from a wide range of fish species, not only in fresh but also in marine and brackish waters. Phylogenetic studies have identified four major genotypes, with a strong geographical relationship. In this study, we have designed and validated a new procedure – named binary multiplex RT ‐ qPCR (bm RT ‐ qPCR ) – for simultaneous detection and typing of all four genotypes of VHSV by real‐time RT ‐ PCR based on dual‐labelled probes and composed by two multiplex systems designed for European and American/Asiatic isolates, respectively, using a combination of three different fluorophores. The specificity of the procedure was assessed by including a panel of 81 VHSV isolates covering all known genotypes and subtypes of the virus, and tissue material from experimentally infected rainbow trout, resulting in a correct detection and typing of all strains. The analytical sensitivity was evaluated in a comparative assay with titration in cell culture, observing that both methods provided similar limits of detection. The proposed method can be a powerful tool for epidemiological analysis of VHSV by genotyping unknown samples within a few hours.