Evaluation and application of eDNA metabarcoding primers of fish: A case study in an estuarine ecosystem

The selection of primers is crucial for conducting eDNA metabarcoding studies, particularly in estuarine regions characterized by complex habitats and high fish diversity. However, most previous eDNA metabarcoding studies have not systematically screened or evaluated primers specifically tailored to the target areas. This study focuses on a high-diversity estuarine region (the Humen Estuary of the Pearl River) to conduct an ecological investigation using eDNA metabarcoding. The results demonstrate that in silico PCR can assist in primer selection for metabarcoding, but practical surveys still require empirical validation through actual water samples. Additionally, by constructing mock communities using fish tissue-derived DNA, this study identified a positive correlation between fish DNA concentration and metabarcoding read abundance. Finally, our findings confirm that the MiFish-U primer set is suitable for investigating fish diversity in the Humen Estuary of the Pearl River.