Finding an Environmental DNA Needle in a Haystack of Congenerics and Inhibitors

Environmental DNA (eDNA) promises to be a powerful tool for sampling a wide range of organisms. However, a particular challenge of eDNA is that the target DNA sequence is always a minority component of the sample. Reliable detection requires that sample analysis overcomes PCR inhibitors, which are ubiquitous in the environment. It also requires an analysis which can distinguish the target from what may be very closely-related non-targets, without resulting in false positives (mistaking a non-target DNA fragment for the target) or false negatives (inability to detect a rare target when non-target sequences are common). Here, we provide a review of considerations when applying quantitative PCR (qPCR) for eDNA analysis, including measuring and controlling PCR inhibition and design of highly specific assays for analysis of complex templates.