1. A novel multiplex RT-qPCR method based on dual-labelled probes suitable for typing all known genotypes of viral haemorrhagic septicaemia virus.
- Author
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Vázquez D, López-Vázquez C, Skall HF, Mikkelsen SS, Olesen NJ, and Dopazo CP
- Subjects
- Animals, Genotype, Novirhabdovirus classification, Oncorhynchus mykiss virology, Sensitivity and Specificity, Hemorrhagic Septicemia, Viral virology, Molecular Typing methods, Novirhabdovirus genetics, Real-Time Polymerase Chain Reaction
- Abstract
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 (bmRT-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., (© 2015 John Wiley & Sons Ltd.)
- Published
- 2016
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