Development and validation of a multiplex quantitative polymerase chain reaction assay for detecting and genotyping red sea bream iridoviral disease

Megalocytiviruses are responsible for severe mortality in various marine and freshwater fish species worldwide. Specifically, the red sea bream iridovirus (RSIV) and infectious spleen and kidney necrosis virus (ISKNV) genotypes are known to cause the red sea bream iridoviral disease (RSIVD). Although several quantitative polymerase chain reaction (qPCR) assays have been developed for diagnosing RSIVD, these methods fail to differentiate between viral genotypes within Megalocytivirus. The present study introduces an innovative multiplex qPCR assay for the precise detection of RSIVD. This diagnostic method targets the histidine triad motif (HIT)-like protein and ORF080L genes, which are unique markers for the RSIV and ISKNV genotypes. The assay exhibited high analytical sensitivity, expressed as the 95% limit of detection (LoD95%), of 8.69 copies/5 mu L for RSIV and 19.07 copies/5 mu L for ISKNV. Notably, the assay exhibited no cross-reactivity and was unaffected by common inhibitory substances. The diagnostic sensitivity and specificity were 100% for both RSIV and ISKNV, demonstrating the reliability of the assay. The multiplex qPCR assay was 100-1000 times more sensitive than conventional PCR methods, emphasizing its importance in early-stage infection detection. The repeatability and reproducibility of the assay were confirmed through extensive testing, showing low variation and high correlation across different technicians and laboratory settings. Furthermore, the ability of the assay to selectively detect and differentiate between RSIVD genotypes represents a significant advancement in aquatic animal disease diagnostics. This multiplex qPCR assay offers a rapid, specific, sensitive, and quantitative diagnostic tool for global RSIVD surveillance efforts and contributes to the prevention and understanding of disease progression in the aquaculture industry.