Detection of Aeromonas hydrophila by Basic and Fluorescent MIRA Assays

<i>Aeromonas hydrophila</i> is a prevalent opportunistic pathogen in aquaculture. To establish a rapid, convenient, and accurate detection method for <i>A. hydrophila</i>, this study developed and evaluated Multi-Enzyme Isothermal Rapid Amplification (MIRA) assays, which could complete amplification within 20 min at a constant temperature of 39 °C. The basic MIRA assay targeting the aerolysin (<i>aerA</i>) gene demonstrated high specificity, showing no cross-reactivity with six related bacterial species including <i>Aeromonas veronii</i>, <i>Vibrio harveyi</i>, <i>Pseudomonas fluorescens</i>, <i>Bacillus subtilis</i>, <i>Bacillus cereus</i>, and <i>Lactiplantibacillus plantarum</i>. The fluorescent MIRA assay achieved a detection limit of 1 fg/μL (3.1 × 10² copies/μL) when using the pUC57-aerA standard plasmid, while real-time quantitative PCR achieved a detection limit of 0.1 fg/μL (31 copies/μL). Thus, the MIRA assay exhibited 10-fold lower sensitivity than qPCR but shortened the reaction time from several hours (nearly two hours) to within one hour. Both the specificity and sensitivity of the MIRA reactions were evaluated with three independent experiments. These findings suggested that the developed MIRA assays provide a rapid, specific, and practical diagnostic tool for <i>A. hydrophila</i> detection in aquaculture environments, particularly suitable for resource-limited field applications.