Development and validation of hydrolysis assays for seven species of exotic fish

Abstract

Determining the distribution and presence of exotic taxa are significant challenges for eradication efforts and for restoration ecology. One method is detect the presence of introduced species that has shown promise is the use of environmental DNA-DNA obtained from an environmental sample such as soil or sediment rather than directly from a tissue sample. As part of Intermediate Output 2 (IO2) of the Lake Ecosystem Restoration New Zealand project, we were asked to develop hydrolysis assays (Commonly called Taqman assays) to detect the presence of seven species of introduced fish. Here we report the sequences of the primers and the primers and the hydrolysis probe for assays for catfish, Ameiurus nebulosus; mosquito fish, Gambusia affinis; goldfish, Carassius auratus; Koi carp, Cyprinus carpio; perch Perca fluviatilis; rudd, Scardinius erythrophthalmus and tench, Tinca tinca. We identified assay conditions that resulted in true-negative results by testing for cross reactivity against the six species of exotic fish that were not the target species. Additionally, we compared the effect of adding different amounts of DNA to the found that adding more DNA template can result in false-negative results (DNA present but not detected) possibly due to the concentration of inhibitory substances reaching concentrations high enough to inhibit the assays.