First generic one step real-time Taqman RT-PCR targeting the RNA1 of betanodaviruses

•A new RT-PCR-based test was developed to detect the RNA1 component of betanodaviruses for diagnostics.•The test is performed in one step by real-time RT-PCR, which reduces the risk of contamination and simplifies the procedure.•The assay is specific, repeatable, robust and can detect 50–100 copies of a plasmid containing the targeted sequence.•The test has been validated on a large range of viral genotypes.

The detection of betanodavirus genomic components is a major issue for diagnostics and control of viral nervous necrosis (VNN), a devastating disease affecting fish worldwide. Despite a number of published molecular-based tests, most of them targeting the RNA2 molecule of the virus, diagnostics is still a challenge due to the high genetic diversity within this genus. In the present study, a new one-step real-time RT-PCR (rRT-PCR), targeting RNA1 of most genotypes of betanodaviruses, was proposed and validated. The test detected successfully various isolates of betanodavirus representatives of the four species RGNNV, SJNNV, TPNNV and BFNNV, either produced on cell culture or from clinical samples. It was specific as shown by the absence of signal on samples from healthy sea bass or from field samples of six other fish species without clinical signs of VNN. The assay detected reliably 50–100 copies of plasmids containing the targeted cloned RNA1 region, as well as an infectious dose of virus of 102.5–102.85 TCID50/ml. A set of samples was tested by two different laboratories, with similar results, demonstrating the robustness of the test. This is the first one step generic rRT-PCR method for betanodaviruses. It is simple to perform and may be used for first intention diagnostics as well as for confirmation in case of doubtful results obtained with other published tests targeting RNA2.