Development and validation of versatile species-specific primer assays for eDNA monitoring and authentication of 10 commercially important Peruvian marine species

Molecular identification assays provide crucial support in the research and regulation of aquatic resources. Among them, species-specific primers provide strong discriminatory power for fast and simultaneous differentiation between closely related species. In this study, we used interspecific variations detected in two mitochondrial genes to develop species-specific primers for eDNA monitoring and identifying 10 fish and shellfish species commercially available within the Peruvian seafood sector. To ensure versatility and high specificity, our primers were subjected to PCR, qPCR, and sequencing methods, coupled with robust validation assays that included a) an in-silico stage using self-generated and public DNA sequences, b) an in-vitro stage using target species belonging to vouchered specimens, fresh and cooked commercial samples, early life stages, and broad taxa of non-target species, and c) an in-situ stage using eDNA samples from different Peruvian marine ecosystems. Our novel species-specific primers successfully passed the validation process with high efficiency and specificity in unequivocally identifying all target species with 100% accuracy and without cross-species amplifications, thereby making them valuable tools for eDNA monitoring, seafood authentication, and to identify and combat illegal, unreported and unregulated fishing. The identification assays presented herein can be used to support effective fishery management and conservation efforts not only in the Peruvian fishery sector but also in other countries where our target species also occur or are available as imported commodities.