Development of a Real-Time PCR Assay for Noninvasive Detection of Miamiensis avidus 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.