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.

Design and Evaluation of a Macroarray for Detection, Identification, and Typing of Viral Hemorrhagic Septicemia Virus (VHSV)

“…Since the macroarray is based on a previously reported PCR amplification procedure validated against a large number of VHSV and non-VHSV rhabdoviruses [14], the number of VHSV strains employed in the present study was limited, focused to ensure that that reliability of the array was not affected. The reference VHSV viral strains used were FR-07-71 (Genogroup Ia; [25]), DK-1p49 (Genogroup II; [1]), MLA98/6WH1 (G. III; [26]), US-MAKAH (G. IVa; [27]), Goby 1-5 (G. IVb; [28]).…”

“…The PCR amplification step of this macroarray is on in the binary multiplex RT-qPCR (bmRT-qPCR) for the diagnosis and typing of VHSV strains of the 4 genogroups, designed and validated by Vázquez et al [14]. In this procedure, the system consists of 2 PCR tubes, MSI and MSII (multiplex systems I and II).…”

“…MSII uses 2 sets of 2 primers and 1 probe; one is specific for genogroup IVa (FAM labelled), and the second one for genotype IVb (HEX labelled). The specificity of the binary multiplex system was successfully evaluated against a panel of 79 VHSV strains of all genogroups and sublineages [14].…”

“…In fact, genotyping is accepted as an important tool for tracing isolates in molecular epidemiology [12]. To this regard, our group has recently reported reverse transcription real-time quantitative PCR (RT-qPCR) procedures for diagnosis and quantification [13] and for typing [14] of this virus. Since those procedures were based on TaqMan ® probes, we considered the possibility of adapting them to the array technology.…”

“…In the study cited above [14], we reported the validation of a binary multiplex RT-qPCR (bmRT-qPCR) for the diagnosis and typing of VHSV strains from any genotype. Therefore, we used that method to design an RT-qPCR based macroarray for the diagnosis and typing of VHSV.…”