An evaluation of target specificity and sensitivity of three qPCR assays for detecting environmental DNA from Northern Pike (Esox lucius)

We developed and evaluated three qPCR assays for detecting environmental DNA from northern pike (Esox lucius). The assays target the cytochrome oxidase 1 (EluCOI), control region (EluCR), and cytochrome b (EluCytB) genes of the mitochondrial DNA. Target specificity, assessed using the fluorescence signal (at 45 cycles) to noise (at 1 cycle) ratio (S/N), showed strong amplification in northern pike (mean S/N = 2.62, 3.52, 2.69 for EluCOI, EluCR, EluCytB). The mean S/N estimates from fifteen non-esocid freshwater fishes were about 1.0, as expected for no amplification. EluCR showed evidence of amplification (mean S/N = 3.16) in muskellunge (Esox masquinongy). The sensitivity tests indicated EluCOI has a higher detection probability than EluCR and EluCytB at low (20 copies/reaction) copy number. The results favor using EluCOI although EluCytB and EluCR are viable assays for the detection of northern pike eDNA.