Robust Detection of Rare Species Using Environmental DNA: The Importance of Primer Specificity
Citation
Wilcox TM, McKelvey KS, Young MK, Jane SF, Lowe WH, et al. (2013) Robust Detection of Rare Species Using Environmental DNA: The Importance of
Primer Specificity. PLoS ONE 8(3): e59520. doi:10.1371/journal.pone.0059520
Summary
Abstract Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probebased chemistries may represent a particularly powerful tool because of the method’s sensitivity, specificity, and potential to quantify target DNA. However, there has been little work understanding the performance of these assays in the presence of closely related, sympatric taxa. If related species cause any cross-amplification or interference, false positives and negatives may be generated. These errors can be disastrous if false positives lead to overestimate the abundance of an endangered species or if false negatives prevent detection of an invasive species. In this study we test factors that influence [...]
Summary
Abstract
Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probebased
chemistries may represent a particularly powerful tool because of the method’s sensitivity, specificity, and potential
to quantify target DNA. However, there has been little work understanding the performance of these assays in the presence
of closely related, sympatric taxa. If related species cause any cross-amplification or interference, false positives and
negatives may be generated. These errors can be disastrous if false positives lead to overestimate the abundance of an
endangered species or if false negatives prevent detection of an invasive species. In this study we test factors that influence
the specificity and sensitivity of TaqMan MGB assays using co-occurring, closely related brook trout (Salvelinus fontinalis)
and bull trout (S. confluentus) as a case study. We found qPCR to be substantially more sensitive than traditional PCR, with
a high probability of detection at concentrations as low as 0.5 target copies/ml. We also found that number and placement
of base pair mismatches between the Taqman MGB assay and non-target templates was important to target specificity, and
that specificity was most influenced by base pair mismatches in the primers, rather than in the probe. We found that
insufficient specificity can result in both false positive and false negative results, particularly in the presence of abundant
related species. Our results highlight the utility of qPCR as a highly sensitive eDNA tool, and underscore the importance of
careful assay design.
