| Summary: | <i>Saprolegnia parasitica</i> induces heavy mortality in aquaculture. The detection of <i>S. parasitica</i> is often time consuming and uncertain, making it difficult to manage the disease. We validated a previously published real-time quantitative PCR (qPCR) assay to confirm the presence of <i>S. parasitica</i> in fish and in water using environmental DNA (eDNA) quantification. Analytical sensitivity and specificity of the assay was assessed in silico, in vitro and the qPCR assay was compared with microbiological cultivation methods to detect and quantify <i>S. parasitica</i> in water samples from a controlled fish exposure experiment and from fish farms. Furthermore, we compared the use of an agar cultivation method and the qPCR assay to detect <i>S. parasitica</i> directly from mucus samples taken from the fish surface. The analytical sensitivity and specificity of the qPCR assay were high. The qPCR assay detected 100% of <i>S. parasitica</i>-positive water samples. In a field study, the qPCR assay and a microwell plate (MWP) enumeration method correlated significantly. Furthermore, the qPCR assay could be used to confirm the presence of <i>S. parasitica</i> in skin mucus. Thus, the qPCR assay could complement diagnostic methods in specifically detecting saprolegniosis in fish and used as a surveillance method for <i>S. parasitica</i> pathogen in aquaculture environments.
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