Validation of a novel one-step reverse transcription polymerase chain reaction method for detecting viral haemorrhagic septicaemia virus

Hyoung Jun Kim, Argelia Cuenca, Niels Jørgen Olesen*

*Corresponding author for this work

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    Viral haemorrhagic septicaemia (VHS) is one of the most serious viral diseases in salmonid and olive flounder farms. Various diagnostic methods for detecting VHS virus (VHSV) are described in the VHS chapter of the World Organization for Animal Health (OIE) Aquatic Diagnostic Manual. A conventional reverse transcription-PCR (cRT-PCR) targeting the viral nucleocapsid gene is recommended for the detection of VHSV and, to some extent, for genotypic classification. However, the recommended assay exhibits low sensitivity for the detection of VHSV genotype IVa isolates and often shows non-specific amplicons when the RNA template is extracted from non-infected fish cell lines. For these reasons, it is necessary to develop a new RT-PCR method for the foolproof detection of all VHSV genotypes and elimination of non-specific results. In this study, we selected five candidate primer sets that target the VHSV nucleoprotein (N) gene, and selected the most sensitive among them (3F/2R). We then established the optimal reaction conditions for these primers, and ensured that no non-specific amplification had occurred in the fish tissues, fish cell lines, or heterologous viruses. The analytical sensitivity of the novel cRT-PCR was compared to that of cell culture assays, real-time RT-PCR, and other cRT-PCR methods and was found to be as sensitive as or superior to the other methods for detecting all VHSV genotypes. Our newly developed cRT-PCR assay was tested with 80 isolates, representing a collection of all known VHSV genotypes worldwide. Clear and unique amplicons were amplified from all 80 VHSV isolates. The reproducibility, and partly the robustness, of the assay were confirmed by an inter-laboratory proficiency tests including nine laboratories. A high diagnostic sensitivity and specificity was confirmed on tissue material from affected fish. In conclusion a highly robust, sensitive and specific cRT-PCR for detection of VHSV was developed and validated.

    Original languageEnglish
    JournalAquaculture
    Volume492
    Pages (from-to)170-183
    ISSN0044-8486
    DOIs
    Publication statusPublished - 1 Jul 2018

    Keywords

    • Cell culture
    • Conventional RT-PCR
    • Novel primer design
    • Real-time RT-PCR
    • Validation method
    • Viral haemorrhagic septicaemia virus

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