Development of a rapid and visual ERA-LFD assay for sensitive detection of Ichthyophthirius multifiliis with...

Ichthyophthirius multifiliis is a highly pathogenic ciliate responsible for "white spot disease" in freshwater fish, causing significant economic losses to the global aquaculture industry. Early and accurate detection of this parasite is critical for disease control, however, current methods often require specialized equipment and trained personnel, limiting their applicability in resource-limited settings. In this study, a rapid, sensitive, and visual detection method for I. multifiliis was developed by combining enzymatic recombinase amplification (ERA) with lateral flow dipstick (LFD) technology. Specific primers and an internal probe targeting the mitochondrial cytochrome oxidase subunit I (COI) gene were designed. After optimizing the reaction conditions, the ERA-LFD assay achieved optimal performance at 43 ℃ for 18 min, with a detection limit of 10⁰ copies/μL of recombinant plasmid DNA and the ability to detect as few as a single theront. The assay exhibited high specificity, showing no cross-reactivity with seven other common fish parasites (Argulus sp., Chilodonella uncinata, Cryptocaryon irritans, Trichodina sp., Gyrodactylus kobayashii, Trypanosoma sp., and Dactylogyrus sp.). Validation using experimentally infected goldfish (Carassius auratus) demonstrated 100% diagnostic concordance for both fish tissue and water samples. This ERA-LFD method combines isothermal amplification with visual readout, eliminating the need for thermal cyclers and specialized personnel. Its simplicity, speed, and high sensitivity make it a promising point-of-care testing (POCT) tool for early warning and surveillance of ichthyophthiriasis in aquaculture.