Development of a Real-Time Enzymatic Recombinase Amplification Assay (RT-ERA) and an ERA Combined with a Lateral Flow Dipstick (LFD) Assay (ERA-LFD) for Enteric Microsporidian (Enterospora epinepheli) in Grouper Fishes

<i>Enterospora epinepheli</i> poses a severe threat to grouper aquaculture due to the absence of effective prevention and treatment strategies. To address this challenge, we developed and validated two isothermal diagnostic tools, the real-time enzymatic recombinase amplification (RT-ERA) assay and the enzymatic recombinase amplification combined with a lateral flow dipstick (ERA-LFD) assay, targeting the 18S rDNA gene of the parasite. These assays operate under isothermal conditions at ≤40 °C and offer rapid detection, with RT-ERA yielding results in 14~20 min and ERA-LFD in approximately 10 min. The RT-ERA assay demonstrated a strong linear relationship between plasmid copy numbers and cycle threshold (Ct) values (y = -2.1226x + 19.562, R² = 0.9915), enabling accurate quantification. Both methods displayed a detection limit of 2 × 10⁰ copies/μL and no cross-reactivity with other aquaculture pathogens. Validation using grouper tissue and water samples from Hainan, China, demonstrated 100% concordance rates with basic ERA and outperformed compared to conventional PCR. These assays provide sensitive, specific, and rapid detection tools for effective monitoring and pathogen load assessment of <i>E. epinepheli</i>, with broad applicability to pathogen detection in aquaculture systems.