Development and validation of a new SYBR green-based qPCR assay for the broad detection of Novirhabdovirus salmonid (IHNV) genotypes

Abstract

Infectious Haematopoietic Necrosis Virus (IHNV) is a WOAH-notifiable pathogen causing severe economic losses in global salmonid aquaculture. While the WOAH-recommended TaqMan assay is the current standard, its diagnostic efficacy is frequently compromised by high execution costs and reduced sensitivity toward specific genotypes, particularly the JS lineage, due to single nucleotide polymorphisms (SNPs) in the probe-binding region. To address these limitations, this study developed a novel SYBR Green-based qPCR assay targeting the highly conserved nucleoprotein (N) gene. Meticulous thermodynamics of a new primer set was optimized by balancing GC content, melting temperature (Tm), and Gibbs free energy (ΔG)—to ensure broad genotype in- clusivity (U, M, E, JN, and JS) while eliminating non-specific amplifications and primer-dimers. The assay demonstrated superior analytical performance, particularly for the JS genotype, yielding a significantly lower Cq value (16.69) compared to the WOAH assay (20.30), which suffered from probe-mismatch issues. The estimated LoD95% was 16.92 copies/reaction, with 100% diagnostic sensitivity and specificity in experimentally infected samples. Validation using 123 field samples from Republic of Korea and Poland revealed that the new assay identified eight IHNV-positive domestic samples initially missed by the WOAH-recommended method, all of which were subsequently confirmed via sequencing. With a coefficient of variation (CV) consistently below 5% and proven inter-laboratory reproducibility across seven facilities, this assay provides a robust, highly sensitive, and cost-effective alternative to probe-based methodologies. It is suited for large-scale surveillance and routine diagnostics, especially in resource-limited settings where detecting diverse viral mutations is critical.