A species-specific digital PCR assay for the endangered blue racer (Coluber constrictor foxii) in Canada

Abstract

The only population of the endangered blue racer (Coluber constrictor foxii) in Canada occurs on Pelee Island, Ontario. The species is threatened by multiple factors including habitat degradation and loss, road mortality, persecution, and potentially predation. We designed and evaluated the performance of an eDNA ddPCR assay that can be used for multiple facets of conservation of this species. We tested the assay in silico and in vitro using DNA of blue racers and co-occurring snake species, and estimated the LOD and LOQ using synthetic DNA. As wild turkey predation has been suggested to negatively affect racers, we tested the assay on eight wild turkey fecal samples. Our assay is specific, can detect the target species at very low levels of concentration (0.002 copies/μL) and can accurately quantify the copy number ≥0.26 copies/μL. We detected no racer DNA in any wild turkey fecal sample. More fecal samples collected at strategic locations during snake peak activity would enable a more thorough assessment of the possibility of turkey predation. Our assay should be effective for other environmental samples and can be used for investigating other factors negatively affecting blue racers, for example helping to quantify blue racer habitat suitability and site occupancy.