Dataset Information

Development and validation of rapid environmental DNA (eDNA) detection methods for bog turtle (Glyptemys muhlenbergii).

ABSTRACT: Bog turtles (Glyptemys muhlenbergii) are listed as Species of Greatest Conservation Need (SGCN) for wildlife action plans in every state it occurs and multi-state efforts are underway to better characterize extant populations and prioritize restoration efforts. However, traditional sampling methods can be ineffective due to the turtle's wetland habitat, small size, and burrowing nature. Molecular methods, such as qPCR, provide the ability to overcome this challenge by effectively quantifying minute amounts of turtle DNA left behind in its environment (eDNA). Developing such methods for bog turtles has proved difficult partly because of the high sequence similarity between bog turtles and closely-related, cohabitating species, most often wood turtles (Glyptemys insculpta). Additionally, substrates containing bog turtle eDNA are often rich in organics or other substances that frequently inhibit both DNA extraction and qPCR amplification. Here, we describe the development and validation of a qPCR assay, BT3, targeting the mitochondrial cytochrome oxidase I gene that correctly identifies bog turtles with 100% specificity and sensitivity when tested on 201 blood samples collected from six species over a wide geographic range. We also developed a full-process internal control employing a genetically modified strain of Caenorhabditis elegans to improve DNA extraction methods, limit false negative results due to qPCR inhibition, and measure total DNA recovery from each sample. Using the internal control, we found that DNA recovery varied by over an order of magnitude between samples and likely explains the lack of bog turtle detection in some cases. Methods presented herein are highly-specific and may offer a more cost effective, non-invasive tool to supplement bog turtle population assessments in the Eastern United States. Poor or differential DNA recovery, which remains unmeasured in the vast majority of eDNA studies, significantly reduced the ability to detect bog turtle in their natural environment.

SUBMITTER: Kirtane AA

PROVIDER: S-EPMC6855662 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Development and validation of rapid environmental DNA (eDNA) detection methods for bog turtle (Glyptemys muhlenbergii).

Kirtane Anish A AA Wilder Maxwell L ML Green Hyatt C HC

PloS one 20191114 11

Bog turtles (Glyptemys muhlenbergii) are listed as Species of Greatest Conservation Need (SGCN) for wildlife action plans in every state it occurs and multi-state efforts are underway to better characterize extant populations and prioritize restoration efforts. However, traditional sampling methods can be ineffective due to the turtle's wetland habitat, small size, and burrowing nature. Molecular methods, such as qPCR, provide the ability to overcome this challenge by effectively quantifying min ...[more]

PMID: 31725720

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Project description:The environmental DNA (eDNA) method is the practice of collecting environmental samples and analyzing them for the presence of a genetic marker specific to a target species. Little is known about the sensitivity of the eDNA method. Sensitivity is the probability that the target marker will be detected if it is present in the water body. Methods and tools are needed to assess the sensitivity of sampling protocols, design eDNA surveys, and interpret survey results. In this study, the sensitivity of the eDNA method is modeled as a function of ambient target marker concentration. The model accounts for five steps of sample collection and analysis, including: 1) collection of a filtered water sample from the source; 2) extraction of DNA from the filter and isolation in a purified elution; 3) removal of aliquots from the elution for use in the polymerase chain reaction (PCR) assay; 4) PCR; and 5) genetic sequencing. The model is applicable to any target species. For demonstration purposes, the model is parameterized for bighead carp (Hypophthalmichthys nobilis) and silver carp (H. molitrix) assuming sampling protocols used in the Chicago Area Waterway System (CAWS). Simulation results show that eDNA surveys have a high false negative rate at low concentrations of the genetic marker. This is attributed to processing of water samples and division of the extraction elution in preparation for the PCR assay. Increases in field survey sensitivity can be achieved by increasing sample volume, sample number, and PCR replicates. Increasing sample volume yields the greatest increase in sensitivity. It is recommended that investigators estimate and communicate the sensitivity of eDNA surveys to help facilitate interpretation of eDNA survey results. In the absence of such information, it is difficult to evaluate the results of surveys in which no water samples test positive for the target marker. It is also recommended that invasive species managers articulate concentration-based sensitivity objectives for eDNA surveys. In the absence of such information, it is difficult to design appropriate sampling protocols. The model provides insights into how sampling protocols can be designed or modified to achieve these sensitivity objectives.

Project description:BackgroundQuantitative polymerase chain reaction (qPCR) assays used in pathogen detection require rigorous methods development including characterizing DNA extraction products. A DNA extract characterization process is demonstrated using DNA extracted from five different cells types (two Gram-negatives: Escherichia coli, and Burkholderia thailandensis, spores and vegetative cells from the Gram-positive Bacillus cereus, and yeast Saccharomyces cerevisiae) with six different methods.ResultsDNA extract quantity (concentration and extraction efficiency) and quality (purity and intactness) varied by cell type and extraction method enabling the demonstration of different DNA characterization methods. DNA purity was measured using UV spectroscopy, where the A260/A280 and A260/A230 ratios are indicators of different contaminants. Reproducibility of UV spectroscopy measurements decreased for DNA concentrations less than 17.5 ng/?L. Forty-seven extracts had concentrations greater than 17.5 ng/?L, 25 had A260/A280 above 2.0, and 28 had A260/A230 ratios below 1.8 indicating RNA and polysaccharide contamination respectively. Based on a qPCR inhibition assay the contaminants did not inhibit PCR. Extract intactness was evaluated using microfluidic gel electrophoresis. Thirty-five samples had concentrations above the limit of quantification (LOQ, roughly 11 ng/ ?L), 93.5% of the DNA was larger than 1kb and 1% was smaller than 300 bp. Extract concentrations ranged from 1502.2 ng/?L to below the LOQ when UV spectroscopy, fluorometry, and qPCR were used. LOQ for UV spectroscopic and fluorometric measurements were 3.5 ng/?L and 0.25 ng/?L respectively. The qPCR LOQ varied by cell type (5.72?×?10-3 ng/?L for E. coli, 2.66?×?10-3 ng/?L, for B. cereus, 3.78?×?10-3 ng/?L for B. thailandensis, and 7.67?×?10-4 ng/?L for S. cerevisiae). A number of samples were below the UV spectroscopy (n?=?27), flurometry (n?=?15), and qPCR (n?=?3) LOQ.ConclusionThe presented DNA extract characterization process provides measures of DNA quantity and quality applicable to microbial detection methods development and validation studies. Evaluating DNA quality and quantity results in a better understanding of process LOD and contributing factors to suboptimal assay performance. The samples used demonstrated the use of different DNA characterization methods presented but did not encompass the full range of DNA extract characteristics.

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Dataset Information

Development and validation of rapid environmental DNA (eDNA) detection methods for bog turtle (Glyptemys muhlenbergii).

Publications

Development and validation of rapid environmental DNA (eDNA) detection methods for bog turtle (Glyptemys muhlenbergii).

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