“Development and validation of a multiplex RT-PCR assay for the simultaneous detection of three major viral pathogens in salmonids, IHNV, ISAV and SAV (7084)”
Written by marina02 on August 20, 2025 . Posted in Abstracts .
Abstract
Development and validation of a multiplex RT-PCR assay for the simultaneous detection of three major viral pathogens in salmonids, IHNV, ISAV and SAV
Infectious hematopoietic necrosis virus (IHNV), infectious salmon anaemia virus (ISAV), and salmon alphavirus (SAV) are significant pathogens responsible for severe infections in salmonid species and are classified as notifiable diseases by the World Organization for Animal Health (WOAH). This study focused on the selection of optimal primer pairs that target highly conserved regions within the genomes of each virus, emphasizing specificity and broad genotype coverage, which were subsequently assessed through single PCR assays. The limit of detection (LOD) for each single PCR was determined to be 1.0×100 copies/µL for IHNV, 1.0×101 copies/µL for ISAV, and 1.0×102 copies/µL for SAV. A multiplex RT-PCR assay was developed to simultaneously detect the three pathogenic salmonid viruses alongside the β-actin gene, which served to validate RNA isolation and cDNA synthesis. The multiplex PCR was optimized to include 0.3 mM dNTPs, 0.5 mM betaine, and GC Melt solution, with an annealing temperature set at 60°C. The LOD for the multiplex PCR was established at 1.0×103 copies/µL for IHNV, 1.0×102 copies/µL for both SAV and ISAV, with a calculated LOD95% of 7.4×102 copies/µL for all viruses. Importantly, the multiplex PCR did not yield non-specific amplification in eight different fish cell lines and four bacterial isolates, with further validation using field samples from aquaculture farms showing no false positives, and robustness evaluations demonstrating consistent and reliable performance across various PCR reagents.
Authors
Ko, Sungjae , Department of Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Presenter Seong, So min , Department of Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Author Kim, Young Chul , Department of Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Author Kim, Kwang Il , Department of Aquatic Life Medicine, Pukyong National University, Busan 48513, Republic of Korea, Author Kwon, Mun Gyeong , Aquatic Disease Control Division, National Fishery Products Quality Management Service (NFQS), Republic of Korea, Author Jeong, Ji-Min , Aquatic Disease Control Division, National Fishery Products Quality Management Service (NFQS), Republic of Korea, Author Hong, Suhee , Department of Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Author