Design, evaluation, and validation of specific primers for accurate detection of VHS virus in rainbow trout ( Oncorhynchus mykiss )

Abstract Accurate detection of Piscine novirhabdovirus (PNRV), also know as viral hemorrhagic septicemia virus (VHSV) in rainbow trout ( Oncorhynchus mykiss ) is essential for effective aquaculture disease management. This study developed strain‐inclusive primers targeting the conserved glycoprotein (G) gene using integrated bioinformatics approaches (NCBI Primer‐BLAST, CLUSTAL W, ALLELE ID, OLIGO). The primer set was designed to detect approximately 200 VHSV strains while maintaining stringent analytical specificity, demonstrating no cross‐reactivity with rainbow trout or human genomic sequences, nor with clinically relevant salmonid pathogens (IPNV, IHNV). Samples from Iranian fish farms underwent standardized molecular processing: RNA extraction, cDNA synthesis, and polymerase chain reaction (PCR) amplification. Conventional PCR consistently identified a 129 bp target fragment, whereas real‐time quantitative PCR demonstrated robust sensitivity across serial cDNA dilutions (1:1 to 1:256). Melt curve analysis and gel electrophoresis confirmed primer specificity and amplification accuracy. Owing to its diagnostic performance, the primer set exhibited functional flexibility across annealing temperatures (53–66°C), enabling optimization for variable sensitivity and strain inclusivity to detect attenuated viral loads and potential genomic variants. This investigation provides a rapid, sensitive, and highly specific molecular diagnostic tool for VHSV surveillance. These findings underscore the imperative for region‐specific diagnostic kits capable of addressing viral genetic heterogeneity and enhancing disease control efficacy in rainbow trout aquaculture systems.