Detection limits of quantitative and digital <scp>PCR</scp> assays and their influence in presence–absence surveys of environmental <scp>DNA</scp>

Hunter, Margaret E.; Dorazio, Robert M.; Butterfield, John S.; Meigs‐Friend, Gaia; Nico, Leo G.; Ferrante, Jason A.

doi:10.1111/1755-0998.12619

Cited by 129 publications

(137 citation statements)

References 45 publications

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“…Incorporating an experimentally derived threshold to remove false positives (as we did here) will also remove true detections, resulting in decreased detection probabilities. Statistical methods for estimating this threshold are also available and can be used to differentiate detections from nondetections (Hunter et al., ). Finally, model‐based methods to account for false positives also exist, and the best option to account for false positives will likely be context dependent.…”

Section: Discussionmentioning

confidence: 99%

Design‐ and model‐based recommendations for detecting and quantifying an amphibian pathogen in environmental samples

Mosher

Huyvaert

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et al. 2017

Ecology and Evolution

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Accurate pathogen detection is essential for developing management strategies to address emerging infectious diseases, an increasingly prominent threat to wildlife. Sampling for free‐living pathogens outside of their hosts has benefits for inference and study efficiency, but is still uncommon. We used a laboratory experiment to evaluate the influences of pathogen concentration, water type, and qPCR inhibitors on the detection and quantification of Batrachochytrium dendrobatidis (Bd) using water filtration. We compared results pre‐ and post‐inhibitor removal, and assessed inferential differences when single versus multiple samples were collected across space or time. We found that qPCR inhibition influenced both Bd detection and quantification in natural water samples, resulting in biased inferences about Bd occurrence and abundance. Biases in occurrence could be mitigated by collecting multiple samples in space or time, but biases in Bd quantification were persistent. Differences in Bd concentration resulted in variation in detection probability, indicating that occupancy modeling could be used to explore factors influencing heterogeneity in Bd abundance among samples, sites, or over time. Our work will influence the design of studies involving amphibian disease dynamics and studies utilizing environmental DNA (eDNA) to understand species distributions.

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Section: Discussionmentioning

confidence: 99%

Design‐ and model‐based recommendations for detecting and quantifying an amphibian pathogen in environmental samples

Mosher

Huyvaert

Chestnut

et al. 2017

Ecology and Evolution

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“…Digital PCR provides sensitive and absolute DNA quantification of target DNA without requiring the use of a standard curve (Nathan, Simmons, Wegleitner, Jerde, & Mahon, ). Digital PCR has some advantages over real‐time qPCR, such as a higher tolerance to PCR inhibitors and a lower possibility of the smaller variations in estimates (Doi, Uchii, et al, ; Hunter et al, ). However, digital PCR requires expensive apparatus and has higher running costs compared with real‐time qPCR.…”

Section: Edna Detectionmentioning

confidence: 99%

The detection of aquatic macroorganisms using environmental DNA analysis—A review of methods for collection, extraction, and detection

et al. 2019

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The field of environmental DNA (eDNA) analysis has rapidly developed over the past decade and the technique has become widely used for detecting aquatic macroorganisms in a variety of habitats. However, a variety of measurement protocols have been individually developed for different eDNA studies and this may lead to confusion for others who wish to incorporate eDNA analysis in their research. It is important therefore to synthesize the current status of—and future challenges to—the methodology of eDNA analysis. We here synthesized the protocols from total 438 published eDNA studies detecting aquatic macroorganisms were used to calculate the frequency of using each method in eDNA analysis steps. We found that the frequency of methods used converged to one or two methods for any analysis step. Furthermore, although the procedure with highest frequency is not always the best, it was shown that the eDNA collection by filtration and subsequent extraction/purification using a DNeasy Blood and Tissue DNA extraction kit (Qiagen, Hilden, Germany) or PowerWater DNA Extraction Kit (Qiagen) is the most common procedure. An understanding of the characteristics of commonly used methods can help those newly engaged in eDNA studies to understand the basic outline of eDNA analysis. Our review will be useful for the future improvement and development of analytical eDNA techniques of eDNA by sharing the recognition of methodological characteristic including advantages and disadvantages in major analytical techniques.

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“…) or optimization of low‐copy DNA detection (e.g., increased biological or technical replicates per sample or qPCR, utilization of more sensitive equipment or methods; Schultz and Lance ; Hunter et al. ) are some of the additional steps that could be tested. Thus, while the challenge of designing, testing, and validating eDNA assays may seem to be a simple list of steps to implement, it is clear that these minimum basic practices can be complex at times and worthy of careful planning and execution in every case.…”

Section: Discussionmentioning

confidence: 99%

Environmental DNA (eDNA) Assays for Invasive Populations of Black Carp in North America

et al. 2019

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The Black Carp Mylopharyngodon piceus is an increasingly widespread invasive species in North America that threatens freshwater mussel populations. We developed four qPCR assays for detecting environmental DNA (eDNA) from these Black Carp populations. Assays were designed to target four mitochondrial DNA loci and were based on 34 complete mitochondrial genome sequences, including 29 generated in this study from samples obtained in three countries. Assays were validated for taxon specificity with in silico comparisons against archived DNA sequences and with in vitro tests of 41 DNA samples from Black Carp, as well as DNA samples from 30 nontarget fish species, all from the Mississippi River basin. All four assays were able to detect the DNA of all Black Carp samples and did not exhibit any positive results with DNA from other tested species. Tests conducted in round‐robin fashion among three different laboratories found that all four assays were able to detect DNA at very low template concentrations (limits of detection = 3 copies/qPCR, limits of quantification = 16–64 copies/qPCR) and, as part of in situ validation, were successful in detecting eDNA from Black Carp in aquaculture ponds. Despite some challenges with other attempts at in situ validation, the assays were also effective in detecting Black Carp eDNA in water samples from a drainage ditch in the upper reaches of the species’ range that was known to contain juvenile Black Carp, as well as in water samples from the Mississippi River and a connected oxbow lake in the lower reaches of the species’ range.

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Detection limits of quantitative and digital PCR assays and their influence in presence–absence surveys of environmental DNA

Cited by 129 publications

References 45 publications

Design‐ and model‐based recommendations for detecting and quantifying an amphibian pathogen in environmental samples

Design‐ and model‐based recommendations for detecting and quantifying an amphibian pathogen in environmental samples

The detection of aquatic macroorganisms using environmental DNA analysis—A review of methods for collection, extraction, and detection

Environmental DNA (eDNA) Assays for Invasive Populations of Black Carp in North America

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