Development of a quantitative PCR assay for detecting northwest salamander (ambystoma gracile) in environmental DNA samples

We developed a primer and probe based quantitative PCR assay for use with environmental DNA to detect Northwest salamander (Ambystoma gracile), a species endemic to the temperate Pacific coastal region of North America. The assay targets a region in the mitochondrial DNA (mtDNA) D-loop gene. Tests of the assay were performed in silico (using the NCBI BLAST tool), in vitro (using DNA extracted from A. gracile and related species) and in situ (water samples collected from a lake with a known population of A. gracile). This assay will be useful in efforts to monitor the distribution and occupancy of this species.