Development and Validation of a Multienzyme Isothermal Rapid Amplification–Based Fluorescence Assay for Detection of Megalocytivirus pagrus 1

Megalocytivirus pagrus 1 is a major viral pathogen that causes high mortality and economic losses in aquaculture worldwide. Although PCR-based diagnostics are highly sensitive and specific, their equipment dependence and long assay times limit field applicability. In this study, a digital multienzyme isothermal rapid amplification with exonuclease probe (digital MIRA-EXO) assay was developed and validated for rapid and specific detection of red sea bream iridovirus (RSIV), infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV). The digital MIRA-EXO assay specifically identified Megalocytivirus pagrus 1 without cross-reactivity with non-target samples, including four fish cell lines and 13 different fish pathogens. Analytical sensitivity, expressed as 95% limit of detection, ranged from 146.87 to 201.6 copies/μL across the three viruses. Diagnostic performance evaluation of 180 fish samples showed high sensitivity (92.22%), specificity (100%), and overall accuracy (96.11%), with an area under the receiver operating characteristic (ROC) curve of 0.971. Substantial agreement with reference assays was observed for experimentally infected (κ = 0.786) and field samples (κ = 0.792), and between column-based and rapid DNA extraction methods (κ = 0.783). Overall, the digital MIRA-EXO assay provides a rapid, accurate, and field-deployable diagnostic tool for Megalocytivirus pagrus 1 detection.