Effective detection of environmental DNA from the invasive American bullfrog

Accurate Detection and Quantification of Seasonal Abundance of American Bullfrog (Lithobates Catesbeianus) Using ddPCR eDNA Assays

“…As the performance of primer/probe assays can be inconsistent between study systems, formal validation, preferentially testing on local natural eDNA samples, should be the rst step in developing large-scale eDNA-based surveillance protocols to ensure that the generated ndings are reliable for the resulting decisions to be pertinent 29,30 . One of the most important aspects that has to be considered when evaluating the performance of a primer/probe assay is its speci city, i.e.…”

“…Nevertheless, in vitro and in situ tests should complement in silico speci city tests, since primers can interact with non-target sequences regardless of nucleotide mismatches 26,51 and the outcome of ampli cation can also signi cantly depend on sample and system speci c PCR conditions 52 . Although primer performance was already comprehensively evaluated in vitro 29,30,50 , we conducted additional in situ tests on natural eDNA samples containing DNA originating from a mixture of sympatric indigenous amphibian species under variable environmental conditions ( Supplementary Table S1), which provides a more pragmatic indication of an assay's speci city in the geographic area of interest relative to an in vitro approach 28 . When both assays were run on water samples collected from bullfrog-free water bodies harboring most of the co-occurring amphibian species in Western Europe, no bullfrog eDNA ampli cation could be observed for either of the two assays under study ( Fig.…”

“…Speci city evaluation is especially important in the context of aquatic eDNA, as its highly degraded nature necessitates the use of shorter sequences as barcodes, which inherently harbors fewer species-speci c regions 19,27 . Although primer speci city of the Goldberg and the Lin assay were previously excessively tested in silico for potential cross-ampli cation with co-occuring non-target species in the Western US and the Beijing area, respectively 29,30,50 , it is imperative to repeat the analyses for amphibian species inhabiting a particular study area. The in silico tests performed here con rmed that both primer/probe assays speci cally detected the bullfrog sequence without interfering with sympatric amphibian species indigenous to Western Europe ( Supplementary Fig.…”

“…Primer/probe assay validation In a rst step, we tested the performance of available primer/probe assays speci cally designed for bullfrog eDNA detection, both in terms of speci city and sensitivity. To the best of our knowledge, three primer pairs targeting bullfrog eDNA were published at the onset of this study, which are referred to as the Ficetola 15 , Strickler 50 , and Lin 30 assays. The Ficetola assay lacks a corresponding probe and was previously shown to have some weaknesses regarding its speci city 30 , which led us to decide not to include this assay for further testing.…”

“…False positives (type I errors), for instance, can emerge from non-speci c binding of primers, whereas de cient primer binding e ciency or non-target template competition, on the other hand, can culminate in false negatives (type II errors) 19,26,27 . Intensively testing the speci city and sensitivity of primer/probe assays in silico, in vitro, and in situ is therefore a crucial, yet frequently undervalued, step to avoid such errors [28][29][30] .…”