Table 1.
Cross-species gene sequence information incorporated into eDNA primer set designa.
Table 2.
Characteristics of PCR primers evaluated in the development of eDNA assays for bullfrog and tailed frog, cross-species plant probe, and a cross-species frog probe.
Table 3.
Effect of technical replicates on binomial standard error of all of the animal qPCR primer sets evaluated on 0.04 μg/L of total DNA isolated from the indicated species.
Fig 1.
Cross-species analysis of qPCR-based detection of mitochondrial gene sequence with primer sets designed towards anurans.
A) Fluorescent-based amplification curves are shown representing assay reactions assembled using TaqMan-associated qPCR primer sets eFrog2, eFrog3, or eFrog5 and total DNA template from bullfrog (LICA), leopard frog (LIPI), tree frog (PSRE), or human (HOSA). Duplicate eDNA qPCR assay reactions are shown. A positive control comprising bullfrog brain cDNA (LICA cDNA) as well as a no template negative control (NTC) were included in the assay. For each reaction, successful amplification of DNA is shown by an increasing fluorescent signal. B) Graphical representation of eFrog3 amplification results of replicate qPCR reactions (n = 23 to 27) performed for against each DNA template with the mean abundance and standard error of the mean shown. Abundance for each qPCR replicate was determined as the assay thermocycle limit (50) minus the cycle threshold (Ct) value when amplification was detected.
Fig 2.
Selectivity in detection following development of species-specific qPCR primer sets.
The species-specific primer sets included eLICA1 (white bar), eLICA2 (grey bar), eASMO (black bar), and eASMO9 (hatched bar). DNA template included in species-specific amplification reactions comprised bullfrog (LICA), leopard frog (LIPI), tree frog (PSRE), clawed frog (XELA), tailed frog (ASMO), and human (HOSA). Additionally, all primer sets were evaluated in a qPCR reaction with no DNA template present (NTC). Multiple qPCR reactions (n = 23 to 27) were performed for each primer set and DNA template combination with the mean abundance and standard error of the mean determined and presented as described in Fig 1.
Fig 3.
Sensitivity of species-specific and animal group qPCR primer sets for (A) bullfrog or (B) tailed frog determined using a concentration range of total DNA. A 5-fold dilution series (0.008, 0.04, 0.2, 1.0 and 5.0 μg/L) of bullfrog frog total DNA was assessed in the qPCR assay against eLICA1 (white triangle), eLICA2 (black triangle), and eFrog3 (white circle) primer sets in Panel A. Panel B shows the results of qPCR assays against eASMO (white square), eASMO9 (black square), and eFrog3 (white circle) primer sets using tailed frog total DNA using the same 5-fold dilution series (0.008, 0.04, 0.2, 1.0 and 5.0 μg/L). The percentage of reactions demonstrating detection following 50 cycles compared to the total reactions performed (n = 23–26 technical replicates) is shown. Negative control reactions containing no DNA template displayed no positive detection score with any of the assays with the sole exception of eFrog3 at 0.04% for the bullfrog template only.
Fig 4.
Examples of the binomial standard error calculated across a range of bullfrog total DNA concentrations (0.008–5.0 μg/L) in a qPCR assay (n = 8 technical replicates) containing the primer sets in Fig 3 for (A) bullfrog and (B) tailed frog.
Fig 5.
Determination of the suitability of environmental samples for eDNA qPCR assessment–Part 1 of a tripartite eDNA assay.
The ePlant5 primer set was used to detect a 147 base pair region of the chloroplast 23S ribosomal RNA gene. Samples were assessed in duplicate and included 5 different environmental water sources (A), municipal tap water (B), human total DNA (C), and distilled water (D). The dashed vertical line denotes the selected data collection cut-off for use in scoring samples based upon the presence of an amplifiable environmental DNA source.
Fig 6.
Schema depicting application of a tripartite test methodology for detection of assayable DNA (circle), animal group (square), and specific species (pentagon) within an environmental water sample to provide greater confidence in eDNA assay results.
*This test result requires additional scrutiny to determine whether the negative animal group outcome is indicating a false positive species-specific detection or not.
Fig 7.
Field validation of eDNA qPCR assays directed towards bullfrog and tailed frog.
Sampling sites were chosen in southern British Columbia (BC), Canada, which included known locations of habitation (star) as well as regions where the target species is absent (circle). Sites selected for validation of the tailed frog eDNA assay were located in the east Kootenay region of southeastern BC. Geographic areas across south Vancouver Island outlined by a dashed line represent locales with historical populations of bullfrog (British Columbia Ministry of Environment, BC Frogwatch Atlas (http://maps.gov.bc.ca/ess/sv/bcfa/) and BullfrogControl.com Inc (http://www.bullfrogcontrol.com/index.html).
Table 4.
Field validation of species-specific eDNA assays directed towards bullfrog and tailed frog.
Table 5.
Stepwise approach in the design, validation, and execution of qPCR-based eDNA assessment.