Amyloodinium ocellatum is one of the most important ectoparasites of marine fish, causing amyloodiniosis and mass mortality in aquaculture. To date, no diagnostic method has been developed to detect and quantify parasite abundance in seawater. In this study, a quantitative real-time PCR (qPCR) assay using the ITS-F3 and ITS-R3 primer pair based on the internal transcribed spacers of nuclear ribosomal DNA (ITS rDNA) of A. ocellatum was successfully established for the detection and quantification of dinospores in seawater. The dinospores were collected by suction filtration of seawater using a vacuum pump, and examination by light microscopy and scanning electron microscopy (SEM) showed that the dinospores were adsorbed on the surface of the cellulose acetate membrane. The linear relationship between seawater parasite abundance and Ct values followed the model y = −3.0607 x + 29.919 with a coefficient of determination (R2) of 0.985. The results of the assay showed high reproducibility and no cross-reactivity with other aquatic pathogens. The assay successfully detected dinospores (100 % success rate) at concentrations of 8 and 10 cell per 300 mL of seawater. The protective fluid buffer effectively protected the dinospores DNA from degradation for over 96 h. The method was used to investigate the life span of dinospores in seawater and the incubation patterns of tomonts in the tank, the results showed that a small fraction of dinospores can survive for more than 7 days in seawater and the process of dinospores incubation from the tomonts persisted up to 84 h. This study provides a valuable quantitative diagnostic tool to assist in the early monitoring and understanding of disease progression in amyloodinosis.
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