Laboratory Validation of a Species-Specific qPCR Assay for Environmental DNA Detection of Senegalese Sole (Solea senegalensis)

The Senegalese sole (Solea senegalensis Kaup, 1858) is a highly valuable species in European aquaculture and capture fisheries. Efficient monitoring of its populations is essential for sustainable management, yet traditional survey methods are invasive and labor-intensive. Environmental DNA (eDNA) analysis offers a powerful non-invasive alternative by detecting DNA fragments shed by organisms into the environment. In this study, we developed a species-specific TaqMan quantitative PCR (qPCR) assay targeting the mitochondrial cytochrome c oxidase subunit I (COI) gene to detect S. senegalensis eDNA. We evaluated assay performance, eDNA release, and persistence under controlled aquarium conditions. The assay demonstrated robust analytical sensitivity and specificity and showed an efficiency of 77.4%, an R2 of 0.986, and an analytical limit of detection at the 10−5 dilution. Density had a statistically significant effect on the values observed (one-way ANOVA: F3,16 = 3.903, p = 0.0287). To ensure accuracy, context-dependent operational quantification cycle (Cq) thresholds were defined: a stringent cutoff (Cq < 30) prevents false positives in complex marine environments, while a permissive cutoff (Cq < 36) maximizes detection in controlled settings. Aquarium trials confirmed that eDNA concentration correlates directly with fish density and accumulation time. Following fish removal, rapid eDNA degradation was observed, falling below strict quantifiable limits within 72 h, although trace signals persisted at late amplification cycles. This optimized qPCR protocol provides a foundational molecular tool that, pending further in situ validation, could support non-invasive S. senegalensis monitoring in ecological assessments and aquaculture management.