RNA Extraction and RT-qPCR for Aquatic Viruses

For the cases investigated, an automated tissue homogenization of samples was performed using the MagNA Lyser instrument (Roche, Basel, Switzerland). Total RNA was extracted using a robot (Roche MagNA Pure LC instrument) with the MagNA Pure LC RNA isolation kit III—Tissue (for the virus), according to the manufacturer’s instructions. The extracted RNA was eluted in 50 μL of nuclease-free water, RNA yields were quantified, and purity was analyzed using the OD260/280 ratio and a NanoPhotometer^® P 300 (Implen, Westlake Village, CA, USA). The eluted RNA was tested immediately following quantitation. The RT-qPCR analysis was done using Light-Cycler 480 RNA Master Hydrolysis Probes for RNA (Roche). For the detection of IPNV, thermocycling conditions and specific primers described by Ingerslev et al. [33 (link)] were used. For piscine orthoreovirus (PRV), the RT-qPCR was done using PRV-specific primers and conditions as described by Palacios et al. [34 (link)]. For the detection of infectious salmon anemia virus (ISAV) in tissue homogenates, ISAV-specific primers and conditions described by Snow et al. [35 ] were used.