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

Abstract

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.