This study evaluates environmental DNA (eDNA) sampling methodologies for monitoring the great crested newt (Triturus cristatus) within Natural England’s District Level Licensing (DLL) scheme areas. Building on previous research, the project compares ethanol precipitation and filtration-based eDNA collection methods, and assesses the utility of DNA metabarcoding alongside species-specific quantitative PCR (qPCR). Ninety ponds across three DLL scheme areas in England were sampled using both methods, with an additional ten field blanks. Filtration samples were analysed using the WC1067 qPCR protocol, and a subset of ten samples underwent 12S metabarcoding to assess biodiversity within these ponds.
Results showed that filtration detected great crested newt eDNA in 41 samples, compared to 33 using ethanol precipitation, with 30 samples testing positive by both methods. Metabarcoding identified 21 species across ten samples, detecting great crested newt in six of eight qPCR-positive samples. However, metabarcoding was less sensitive than qPCR, particularly in samples with low eDNA concentrations. Human DNA contamination was prevalent, potentially masking detection of target species.
For single species testing, the findings are reasonably comparable using filtration and ethanol precipitation, and higher sample volumes could account for the additional samples testing positive by filtration. While metabarcoding offers broader biodiversity insights, its lower sensitivity limits its standalone use for species-specific monitoring.