title: A novel multiplex RT-qPCR method based on dual-labelled probes suitable for typing all known genotypes of viral haemorrhagic septicaemia virus.
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.
authors.affiliation: Aquaculture Institute, Santiago de Compostela University, 15782, Santiago de Compostela, Spain.
authors.forename: D
authors.initials: D
authors.lastname: Vázquez
authors.affiliation: Aquaculture Institute, Santiago de Compostela University, 15782, Santiago de Compostela, Spain.
authors.forename: C
authors.initials: C
authors.lastname: López-Vázquez
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark.
authors.forename: H F
authors.initials: HF
authors.lastname: Skall
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark.
authors.forename: S S
authors.initials: SS
authors.lastname: Mikkelsen
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark.
authors.forename: N J
authors.initials: NJ
authors.lastname: Olesen
authors.affiliation: Aquaculture Institute, Santiago de Compostela University, 15782, Santiago de Compostela, Spain.
authors.forename: C P
authors.initials: CP
authors.lastname: Dopazo
mesh_terms: D000818:Animals; D005838:Genotype; D031941:Hemorrhagic Septicemia, Viral / Q000821:virology*; D058889:Molecular Typing / Q000379:methods*; D029141:Novirhabdovirus / Q000145:classification / Q000235:genetics*; D017686:Oncorhynchus mykiss / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity
keywords: VHSV; multiplex RT-qPCR; typing