Development and Validation of a Multiplex <scp>RT</scp> ‐ <scp>PCR</scp> Assay for the Simultaneous Detection of Three Major Viral Pathogens in Salmonids: <scp>IHNV</scp> , <scp>ISAV</scp> and <scp>SAV</scp>

In this study, a multiplex RT-PCR assay was developed for the simultaneous detection of three major viral pathogens in salmonids: infectious haematopoietic necrosis virus (IHNV), infectious salmon anaemia virus (ISAV) and salmonid alphavirus (SAV). Primer pairs targeting highly conserved regions of each viral genome were designed based on specificity and broad genotype coverage and first validated through single RT-PCR assays. A β-actin gene was included as an internal control to monitor RNA extraction and cDNA synthesis efficiency. The limit of detection (LoD) in single RT-PCR assays was determined as 1.0 × 10⁰ copies/μL for IHNV, 1.0 × 10¹ copies/μL for ISAV and 1.0 × 10² copies/μL for SAV. The multiplex RT-PCR assay was optimised to detect all three viruses and β-actin simultaneously. Compared with WOAH singleplex PCRs, the assay achieved a LoD_95% of 5.9 × 10² copies/μL across targets. It also showed high specificity, with no cross-reactivity to salmonid bacteria and only the β-actin internal control amplified in fish tissues and cell lines. This assay exhibited high specificity in field samples, with IHNV detected in one of the farms tested. Furthermore, it successfully identified IHNV in infected specimens obtained from controlled infection experiments. Through robustness testing, the assay demonstrated consistent performance across all eight reagent kits, thereby confirming its reproducibility within the overall PCR reagent system. This assay offers a rapid, sensitive and specific diagnostic tool for detecting viral infections in salmonid aquaculture.