Nanoprobe-enhanced loop-mediated isothermal amplification detection of Vibrio anguillarum in turbots (Scophthalmus maximus): toward advanced fish disease management

초록

Vibrio anguillarum is a virulent pathogen responsible for severe diseases in mariculture. Its accurate on-site detection remains a challenge due to limitations in established methods, such as insufficient specificity, prolonged assay, and the requirement of non-movable instruments. We developed a visual platform for the rapid, specific, and sensitive detection of V. anguillarum. It integrates loop-mediated isothermal amplification (LAMP) with DNA-functionalized gold nanoparticles (DNA-AuNPs); we termed it "nanoprobe-enhanced LAMP." These DNA-AuNP probes enabled the colorimetric discrimination of empA expression levels within 20 min, while reducing the limit of detection to 1 fg/mu L level (equivalent to 89 CFU/mL). More importantly, this technique demonstrated exceptional specificity in distinguishing V. anguillarum from other Vibrio spp. and foodborne bacteria via selective DNA recognition, outperforming conventional qPCR and fluorescence-based LAMP assays. We also exhibited the applicability of this method by detecting V. anguillarum in various tissues of infected turbots (Scophthalmus maximus), including liver, spleen, kidney, and skin; these results were consistent with the pathological findings. Given its simplicity, portability, and robustness-requiring only a constant temperature source-this approach is ideally suited for field-deployable diagnostics in resource-limited aquaculture settings.

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