Effective detection of environmental DNA from the invasive American bullfrog

Abstract

The American bullfrog (Rana catesbeiana) is one of the most damaging invasive species worldwide. Recently developed environmental DNA (eDNA)-based methods have proven to be promising tools to detect aquatic invasive species at low abundance. To develop an eDNA-based assay for bullfrog monitoring, we designed a primer and probe set that targeted the bullfrog DNA and compared its species specificity in in silico PCR and in vitro quantitative PCR. We demonstrated that our primers successfully amplified the bullfrog DNA, but not DNA of native anuran species in the Beijing area, in silico and in vitro. Additionally, in silico PCR identified only a small number of potentially amplifiable species occurring in North America. In contrast, a previously reported primer pair that specifically targeted the bullfrog in France amplified the DNA of a wide range of teleost fish and amphibians in silico and a common native species in vitro. We also compared three commonly used eDNA extraction methods—the phenol–chloroform–isoamyl alcohol (PCI) method, cetyltrimethylammonium bromide (CTAB)-based extraction, and Qiagen’s DNeasy blood and tissue kit—for their efficiency in recovering bullfrog eDNA from laboratory aquariums. The CTAB method showed a significantly higher extraction efficacy than the PCI method or the DNeasy kit and considerably outperformed the other methods in terms of time efficiency and costs. Our study provides a useful primer set and extraction protocol for eDNA-based active surveillance of bullfrog invasions over a potentially wide geographical range, which has important implications for the management of this invasive species.