Development of a Multienzyme Isothermal Rapid-Amplification Lateral Flow Assay for On-Site Identification of the...

Eel populations are globally threatened by overfishing and illegal trade, making accurate species identification essential for resource conservation and regulatory enforcement. Conventional molecular identification methods are generally applied in the laboratory, with limited rapid on-site application. This study developed a field-deployable assay to identify the Japanese eel (Anguilla japonica), by incorporating multienzyme isothermal rapid amplification (MIRA) technology with a visually readable lateral flow assay (LFA). Species-specific primers targeting a 286 bp region within the mitochondrial genome of A. japonica were designed and labeled with fluorescein amidite and biotin, respectively. The performance of the MIRA-LFA was validated by assessing its specificity against four other major eel species and its analytical sensitivity, i.e., limit of detection (LoD), under optimized temperature and reaction-time conditions. The MIRA-LFA demonstrated 100% specificity, generating a positive signal only for A. japonica, with no cross-reactivity. A clear visual result was obtained within 10 min at the optimal reaction temperature of 39 °C. Under these optimal conditions, the assay showed a high sensitivity, with an LoD of 0.1 ng/μL of genomic DNA. The proposed assay is an effective tool for the rapid, specific, and sensitive identification of A. japonica. The ability to obtain fast, equipment-free visual results makes this assay an ideal point-of-care testing solution to combat seafood fraud and support the sustainable management of this economically important and vulnerable species.