Development of a Real‐Time <scp>PCR</scp> Assay for Noninvasive Detection of <scp><i>Miamiensis avidus</i></scp> in Olive Flounder Aquaculture

Miamiensis avidus (syn. Philasterides dicentrarchi) leads to high mortality and economic losses in olive flounder (Paralichthys olivaceus) aquaculture. In this study, we developed a real-time PCR assay targeting the cytochrome c oxidase subunit I (cox1) gene of M. avidus to sensitively detect and quantify the parasite in seawater. The assay showed a strong linear correlation between the log copy number of the standard plasmid and Ct values (R² = 0.9985), achieving an amplification efficiency of 96.26%. The cox1 gene copy number was estimated at 6017 ± 2794 copies per cell. The assay specifically detected M. avidus (cox1 genotypes 1-4) without cross-reacting with Cryptocaryon irritans or Uronema elegans, identifying as few as two M. avidus cells in 1 L of seawater with 100% accuracy (Ct value: 31.95 ± 0.78). The noninvasive seawater sampling demonstrates higher sensitivity than invasive tissue sampling. A high density of M. avidus (57.81 cells/10 L) was detected in tanks housing olive flounder with clinical signs of infection, and sporadic detection occurred in both inflow and effluent seawater. The developed real-time PCR assay provides a sensitive, specific and noninvasive method for detecting and quantifying M. avidus, enabling improved disease prevention and management in fish farms.