Use of RNase H‐dependent PCR for discrimination and detection of closely related species from environmental DNA

Species‐specific, probe‐based quantitative PCR (qPCR) assays are now commonly used to detect aquatic species from environmental DNA. However, probe‐based qPCR alone does not always provide the specificity needed to distinguish closely related, congeneric species, which may result in amplification of non‐target DNA, causing false positives from eDNA samples. Here, we developed species‐specific qPCR assays using RNase H‐dependent PCR (rhPCR) for detecting closely related fish species from environmental DNA. We found that rhPCR allowed us to achieve specificity that was not possible with TaqMan® qPCR alone, and we used these assays for species detection from eDNA samples. Use of rhPCR will allow species‐specific detection from environmental DNA for a broad range of species including those that occur in sympatry with other closely related, congeneric species, which has not always been possible with probe‐based qPCR alone.