Evaluating an eDNA Survey for an Invasive Amphibian
An environmental team uses quantitative PCR (qPCR) to detect invasive bullfrog DNA in 12 ponds. Each pond is sampled with one 1-L water sample filtered and extracted; each extract is run in three qPCR replicates. Five ponds are positive in at least one replicate. Conventional dip-net surveys find bullfrog tadpoles in two ponds. Pond 7 is positive in all three replicates with Ct 34 (limit of quantification 36; limit of detection 40). Pond 9 is positive in one of three replicates with Ct 38. An extraction blank run alongside the samples amplifies at Ct 39. No positive control fails. The manager wants to know which ponds to treat and whether pond 7 and pond 9 should be considered positive. The team has budget for 20 additional qPCR reactions or 6 site visits.
1. Interpret the qPCR results, including the extraction blank. Should pond 7 and pond 9 be considered positive? What are the likely sources of false-positive and false-negative results?
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Pond 7 is probably positive: all three replicates amplify at Ct 34, below the LOQ of 36, so the target is present but the quantity is uncertain. Pond 9 is equivocal: one replicate at Ct 38 is above the LOQ and close to the extraction blank at Ct 39, so contamination or low-level DNA is possible. The extraction blank indicates trace contamination, so pond 9 should not be treated as positive without resampling. False negatives can arise from DNA degradation, low shedding, dilution, or PCR inhibitors; false positives from contamination, carryover, or non-target amplification. eDNA also does not prove live bullfrogs are present.
2. Design a confirmatory sampling plan using the available budget. Prioritize ponds and explain how you would reduce uncertainty.
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Use the budget to confirm rather than expand prematurely. Prioritize pond 7 and pond 9: collect three independent 1-L samples from each on separate visits, run three qPCR replicates per extract (18 reactions total), and include a field blank and extraction blank (2 reactions) for 20. If possible, pair the visits with dip-net surveys at both ponds. Treat pond 7 as the highest priority because it is consistently positive; pond 9 needs clean resampling to distinguish contamination. Also run dilution tests for inhibition and use multiple negative controls. This design reduces both false-positive and false-negative uncertainty.
3. The manager asks whether eDNA positivity alone justifies eradication treatment. Give a reasoned recommendation.
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eDNA positivity alone does not justify eradication. It shows DNA presence, not necessarily a viable population, and the extraction blank shows contamination risk. Treatment is costly and can harm non-target species. I would recommend treating only ponds confirmed by repeated eDNA plus conventional detection, and using pond 7 as a priority for confirmation because it is consistently positive. Pond 9 should be resampled before any action. Meanwhile, monitor connected ponds and use strict field hygiene to prevent spread.