An Optimised eDNA Protocol for Fish Biodiversity Assessment in a Tropical River System Using PCI Extraction

Environmental DNA (eDNA) analysis has emerged as an innovative and promising method for monitoring aquatic biodiversity, although challenges remain regarding sample filtration and standardisation of extraction protocols. We optimised filtration and extraction techniques using cellulose nitrate (CN) membranes with a 0.45 µm pore size and a phenol–chloroform–isoamyl alcohol (PCI) extraction method. The eDNA collected was quantitatively and qualitatively assessed using a NanoDrop spectrophotometer and agarose gel electrophoresis, respectively. PCR amplification of the 12S rRNA gene was followed by high-throughput metabarcoding to assess fish biodiversity. Our survey identified a diverse aquatic community, uncovering 9 orders, 14 families, 18 genera, and 21 species of fish, with the Cichlidae and Anabantidae families being frequently detected. Dominant species included Oreochromis niloticus and Oreochromis mossambicus. The study concludes that the CN membrane with a 0.45 µm pore size, combined with PCI extraction, offers a cost-effective and reliable protocol, enhancing purity and reducing degradation. This study underscores the feasibility and effectiveness of eDNA analysis in Malaysia, offering a robust framework for future ecological research and conservation efforts in resource-limited settings.