Recognizing false positives: synthetic oligonucleotide controls for environmental DNA surveillance

Environmental DNA (eDNA) is increasingly used for surveillance and detection of species of interest in aquatic and soil samples. A significant risk associated with eDNA methods is potential false positive results due to laboratory contamination. To minimize and quantify this risk, we designed and validated a set of synthetic oligonucleotides for use as species-specific positive PCR controls for several high-profile aquatic invasive species. The controls consist of species-specific sequences for the species of interest, with the addition of a synthetic insert containing recognition sites for several restriction enzymes. Following PCR, the presence of the synthetic insert can be detected using gel electrophoresis, restriction enzyme digests, or DNA sequencing. For quantitative PCR (qPCR), false positives in environmental samples can also be detected using a fluorescent probe designed to detect the synthetic insert. The generation of synthetic controls is a cost-effective, reproducible method that increases the power and reliability of eDNA testing by eliminating misinterpretation of false positive results from laboratory contamination.