Application of Environmental DNA for Inventory and Monitoring of Aquatic Species
@Zerina Rahic
How to Design Species-Specific Primers and Probe for qPCR
- Create an inclusive consensus sequence that incorporates all within-species variability for a species in a well-known region of DNA. Mitochondrial DNA is preferred because it is more abundant than nuclear DNA, and more sequence data are available. Use sequences published in GenBank (National Center for Biotechnology Information, 2012), or sequence tissue samples of target species. It is important that the data incorporated include adequate sampling in the geographic area where the test will be applied.
- For the selected probe chemistry, set appropriate qPCR primer software to design short, unique sequences for use as forward- and reverse-primers and probe. Optimal probe length will differ by chemistry. These primers and probe will allow for amplification and detection of the target sequence (90–120 base-pair length is recommended).
- Compare the resulting design to sequences in GenBank to determine if the sequences are likely to cross-amplify with other species. Try to incorporate as many differences as possible (at least 2 on each primer and 2 on the probe, including 1 toward the 3’ end) between the primer/probe design for your target species and any other species in the database.
Field Sampling
Four methods for field sampling have been developed to date: (1) collect 15 mL of water, preserve using ethanol and sodium acetate, and freeze immediately (Ficetola and others, 2008; Thomsen and others, 2012), (2) filter water through a cellulose nitrate filter (Goldberg and others, 2011), (3) filter water through a glass fiber filter (Jerde and others, 2011), and (4) filter water through carbonate filter (Takahara and others, 2012) (table 1; figs. 2–4). The latter three methods require pumps (either in-line, such as a peristaltic pump, or vacuum-line) and measurement of water filtered (volumes of 1–10 L are common). Filter methods also require either freezing of the filter paper (Jerde and others, 2011; Takahara and others, 2012) or dehydration of the filter paper in vials with molecular-grade ethanol (Goldberg and others, 2011). Although all these methods have been successful, ongoing testing, standardization, and optimization of field and laboratory protocols will continue to improve applications for inventory and monitoring programs.
DNA Extraction and Amplification
DNA extracted from the preserved samples is stable once it has been purified and preserved, and only a portion is used in each PCR reaction. This preserved DNA can be later tested for additional species if desired.
Following DNA extraction, qPCR analysis provides detection information about the target species’ DNA. Although the amount of target DNA present in field samples may be quantified (Thomsen and others, 2012; Takahara and others, 2012), this fact sheet is limited to presence/absence information.
Sources of Error
Identifying sources of error or uncertainty is a critical process in any study, especially for monitoring programs where results could influence future management decisions. Darling and Mahon (2011) provide an excellent overview of potential sources of uncertainty associated with DNA-based methods for monitoring aquatic species. The following points are important when using eDNA methods.
Design of Molecular Assay
Assay design must account for the variation within a species and the variation among species. Failure to incorporate the full range of genetic variation of a target species can lead to false negatives, whereas failure to incorporate the full range of genetic variation in closely related, co-occurring species can lead to false positives. Therefore, it is important to select a genetic region that maximizes the amount of genetic information available for target and related non-target species. For some species, this may require sequencing additional samples to ensure the assay is both sensitive and specific.
Quality Control
Positive and negative controls are necessary to ensure quality and reliability of results at each stage of the study. All DNA extractions should include a negative control, so that cross contamination between extracts can be detected. Each well of the PCR plate should include an internal positive control to ensure that the reaction is not inhibited. All eDNA extractions and qPCR setups should be conducted in a PCR-free laboratory space where concentrated (such as from tissue) DNA samples have not been handled. Thermocyclers and real-time PCR machines should be located outside of this space.
Detection Probability
Like other field-based sampling, results of eDNA detection may have some inaccuracy, and replicate samples are required to estimate occupancy while accounting for uncertainty. In other words, not detecting DNA of a species does not mean it is absent. The lower limits of detection for species are currently unknown and likely vary depending on the species and its density, size, behavior, and habitat.
Field Negative Controls
Two types of negative controls are often employed in the field to increase accuracy. First, samples from a few sites outside the range of a target species are used to confirm non-detection in locations where the species is not present. Second, samples are collected from distilled water using the field protocol at each site to ensure that cross contamination is not occurring between replicate samples within a site and between sites. Sterile gloves, filters, water collection bottles, and sample containers reduce risk of contamination. High-quality sample tubes placed individually inside plastic, sealable bags can reduce cross-contamination should leakage occur when samples are stored or shipped to laboratories.
Timing of Sampling
The timing of sampling may need to coincide with the life history or behavior of a target species. For example, during reproduction when young-of-year are present, eDNA may be abundant. The arrival of migratory species can be detected assuming no other life stages of the species remain in the system.
PCR Replication
Degraded, low-quantity DNA samples are often analyzed in triplicate to ensure detection of DNA (Waits and Paetkau, 2005) and to assess potential false-positives. Using this approach, additional analysis is required if results are not uniform. Standard curves should be developed based on DNA obtained from tissue samples of target species and span the range of sample results.
References
| (Author, year) | Link |
|---|---|
| By David S. Pilliod, Caren S. Goldberg, Matthew B. Laramie, and Lisette P. Waits, 2012 | https://pubs.usgs.gov/fs/2012/3146/ |
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