Development and validation of a quantitative qPCR assay for detecting Natterjack toad ( Epidalea calamita ) eDNA samples

The Natterjack toad ( Epidalea calamita ) is the rarest amphibian species in Ireland, regionally Red-Listed as Endangered. We applied an eDNA approach to detect species presence in breeding pond water samples. We developed a species-specific qPCR assay targeting the cytochrome c oxidase subunit I (COI). The assay was tested in silico, in vitro (DNA extracted from tissue) and in vivo (DNA extracted from water samples). Water samples were collected from five ponds with known Natterjack toad presence or absence to validate the sensitivity and specificity of the assay. The assay was shown to be highly specific to the Natterjack toad and tested positive only against toad tissue samples and eDNA samples from ponds with known species presence. We believe this method can be used for rapid assessment of species occurrence.