Implementation of Novel Design Features for qPCR-Based eDNA Assessment - Fig 4

<p><b>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164907#pone.0164907.g003" target="_blank">Fig 3</a> for (A) bullfrog and (B) tailed frog.</b></p

Selectivity in detection following development of species-specific qPCR primer sets.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164907#pone.0164907.g001" target="_blank">Fig 1</a>.</p

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.

<p>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.</p

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.

<p>*This test result requires additional scrutiny to determine whether the negative animal group outcome is indicating a false positive species-specific detection or not.</p

Field validation of eDNA qPCR assays directed towards bullfrog and tailed frog.

<p>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 (<a href="http://maps.gov.bc.ca/ess/sv/bcfa/" target="_blank">http://maps.gov.bc.ca/ess/sv/bcfa/</a>) and <a href="http://BullfrogControl.com" target="_blank">BullfrogControl.com</a> Inc (<a href="http://www.bullfrogcontrol.com/index.html" target="_blank">http://www.bullfrogcontrol.com/index.html</a>).</p

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.

<p>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.</p

Cross-species analysis of qPCR-based detection of mitochondrial gene sequence with primer sets designed towards anurans.

<p>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 (C_t) value when amplification was detected.</p

Field validation of species-specific eDNA assays directed towards bullfrog and tailed frog.

<p>Field validation of species-specific eDNA assays directed towards bullfrog and tailed frog.</p

Stepwise approach in the design, validation, and execution of qPCR-based eDNA assessment.

<p>Stepwise approach in the design, validation, and execution of qPCR-based eDNA assessment.</p