Rapid Visual Detection of Red Sea Bream Iridovirus Using a Novel Cross-Priming Amplification-Based Lateral Flow Assay

Red sea bream iridovirus (RSIV) occurs mainly at high water temperatures and infects more than 30 different species of fish. In Asia, infected fish cause mass mortality every year. Molecular diagnostics is a technology that efficiently detects and identifies a wide range of fish pathogens through rapid and sensitive analysis of their genetic material. Rapid viral detection is essential for effective disease control. In this study, we developed and validated a cross-priming amplification-based lateral flow assay (CPA-LFA) suitable for field diagnosis owing to its short diagnostic time and simple diagnostic process. The CPA-LFA achieved optimal performance with concentrations of 4 mM MgSO₄, 1.2 mM dNTPs and 0.7 M betaine, with the reaction conducted at 60°C for 60 min. The developed CPA-LFA could specifically identify RSIV without cross-reactivity with several pathogens commonly reported in various fish cell lines and fish. The 95% limit of detection (LOD_95%) of CPA-LFA was 385.76 copies/μL, which was comparable to that of conventional polymerase chain reaction (PCR). Quantitative PCR (qPCR) was used to identify true-positive and true-negative samples from 210 fish samples (160 from cultured fish and 50 from artificially infected fish). Compared with qPCR, CPA-LFA classified six positive samples as false positives. The viral load of these samples was determined to be less than 195.1 copies/μL. The diagnostic sensitivity and specificity of CPA-LFA were 94.34% and 100%, respectively. Furthermore, inter-operator reproducibility testing yielded a kappa value of 1.0, indicating perfect agreement. Therefore, the novel CPA-LFA is especially well-suited for field diagnostics owing to its straightforward diagnostic procedure and capability to quickly and accurately detect RSIV.