Name: development and testing of species-specific quantitative pcr assays for environmental dna applications
Uploaded: 2020-11-05T14:00:00
Duration: 8 min 54 s
Description: Watch this Scientific Journal Video about Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications at JoVE.com

We thank Alvi Wadud and Trudi Frost who helped in primer development and testing. Funding for the assay design reported in this study was provided by the Department of Defense Strategic Environmental Research and Development Program (RC19-1156). Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Data generated during this study are available as a USGS data release https://doi.org/10.5066/P9BIGOS5.

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+considerations+for+the+application+of+environmental+DNA+methods+to+detect+aquatic+species.">Critical considerations for the application of environmental DNA methods to detect aquatic species.</a> Methods in Ecology and Evolution. 7 (11), 1299-1307 (2016).</li><li>Guan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+DNA+(eDNA)+Assays+for+Invasive+Populations+of+Black+Carp+in+North+America.">Environmental DNA (eDNA) Assays for Invasive Populations of Black Carp in North America.</a> Transactions of the American Fisheries Society. 148 (6), 1043-1055 (2019).</li><li>Mosher, B. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freshwater+fisheries+assessment+using+environmental+DNA:+A+primer+on+the+method,+its+potential,+and+shortcomings+as+a+conservation+tool.">Freshwater fisheries assessment using environmental DNA: A primer on the method, its potential, and shortcomings as a conservation tool.</a> Fisheries Research. 197, 60-66 (2018).</li><li>Forootan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+determine+limit+of+detection+and+limit+of+quantification+in+quantitative+real-time+PCR+(qPCR).">Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR).</a> Biomolecular Detection and Quantification. 12, 1-6 (2017).</li><li>Sengupta, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+DNA+for+improved+detection+and+environmental+surveillance+of+schistosomiasis.">Environmental DNA for improved detection and environmental surveillance of schistosomiasis.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (18), 8931-8940 (2019).</li><li>Klymus, K. E., Richter, C. A., Chapman, D. C., Paukert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+eDNA+shedding+rates+from+invasive+bighead+carp+Hypophthalmichthys+nobilis+and+silver+carp+Hypophthalmichthys+molitrix.">Quantification of eDNA shedding rates from invasive bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix.</a> Biological Conservation. 183, 77-84 (2015).</li></ol>

The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

As with any study, defining the question to be addressed is the first step and the design of the eDNA assay depends upon the scope of the study26. For instance, if the goal of the research or survey is to detect one or a few species, a targeted probe-based assay is best. If, however, the goal is to assess a larger suite or assemblage of species, high throughput sequencing metabarcoding assays are better suited. Once it is determined which approach to take, a pilot study including assay design, testing, and optimization is recommended24. Assay design starts with a list of species as described in Figure 1. This list will be the basis for understanding how well an assay performs in terms of specificity and the geographic range it might be applied to6,10. It is encouraged to design the assay for a specific geographic area, enabling the designer to better test an assay for cross-reactivity against other species in that area, and to be aware of the limitations this has on extending an assay to other areas where a target species may occur24. Once the list is complete, sequences can be downloaded from public genetic databases. Since these databases are incomplete27, one should sequence as many species on the list as possible in house to complete the local reference database of sequences that will be used in assay design. Prioritize co-occurring closely related species, as these are the most likely non-targets that will amplify. Focusing on all species within the same genus or family as the target species is a good place to start. Comparisons with closely related species will help identify sequence regions unique to the target species. This can help inform how the assay may perform in other systems or locations. Mitochondrial regions are the usual choice for assay development, because more sequence information from a wider variety of species is available at mitochondrial genes that have been used in barcode of life projects, and because mitochondrial DNA is present at much greater concentration in copies/cell than nuclear DNA24,28,29. Multiple gene regions should be assessed for further assay development as sequence coverage varies among taxa in the genetic repository databases. After this local database of reference sequences is created, a combination of manual visualization of aligned sequence data and computer software programs is used to design the primer/probe assays. One should not rely strictly on software to determine which assays to test. It is important to verify visually on alignments where the primers and probes sit on the targets and non-targets to get a better understanding of how they might act in a PCR. Finally assay screening and optimization includes three levels (in silico, in vitro and in situ)6,7,24,25. In silico design and testing are important for producing a short list of assays with a good chance of success, but empirical (in vitro) testing is crucial for selecting the assay with the best actual performance. In vitro optimization and testing of assays include measuring the reaction efficiency and defining the assay&#8217;s sensitivity and specificity. Limits of detection and quantification are two parameters often overlooked in assay development but important for data interpretation. By running multiple replicates of the standard curves for an assay, LOD and LOQ can easily be measured1,5,30. Few studies discuss results with respect to the assay&#8217;s LOD or LOQ, but Sengupta et al. (2019) incorporate their assay&#8217;s LOD and LOQ into their data interpretation and graphics for a clearer understanding of their results31. Internal positive controls should be multiplexed into the designed assay as well. Without testing for PCR inhibition in the samples, false negatives may occur24,32. We propose the use of a multiplexed IPC assay with the target assay as the easiest method for PCR inhibition testing23. Finally, in situ testing of the assay from field and laboratory collected samples is necessary to ensure target amplification occurs in environmental samples24.
Limitations exist for the use of species-specific, probe-based qPCR assays with eDNA samples. For instance, the design of multiple assays for testing may be limited by sequence availability, and compromise may be necessary on aspects of assay performance. These choices must be guided by the goals of the study and must be reported with the results26. For example, if the goal is detection of a rare species and few positives are expected, an assay with imperfect specificity (i.e., amplification of non-target species) could be used if all detections will be verified by sequencing. If the goal is monitoring the geographic range of a species and eDNA concentration data is not needed, an assay with imperfect efficiency could be used and data reported only as percent detection. Furthermore, unless all potential conspecifics are tested in the laboratory, which is rarely possible, one cannot know with absolute certainty the&#160;true specificity of an assay. For instance, the assay was designed and tested against several freshwater mussel species in the Clinch River. To use this assay in a different river system, we would need to test it against a suite of species in the new location. Genetic variation within the species or population that is not tested during assay development might also affect specificity. Finally, even if an assay has been verified to have high technical performance; conditions change when working in the field. Non-assay related conditions such as water flow, pH, and animal behavior can change eDNA detectability as can use of different eDNA collection and extraction protocols. Using assays that are optimized and well described will help facilitate understanding of the influence such&#160;parameters have on&#160;eDNA detection.
The field of eDNA is maturing beyond the stage of exploratory analysis to increasing standardization of methods and techniques. These developments will improve our understanding of eDNA techniques, abilities, and limitations. The optimization process we outline above improves an assay&#8217;s sensitivity, specificity, and reproducibility. The ultimate goal of this refinement and standardization of eDNA methods is to improve researchers&#8217; abilities to make inferences based on eDNA data as well as increase end-user and stake-holder confidence in results.

Researchers and managers are increasingly becoming interested in the use of environmental DNA assays for species detection. For three decades, quantitative or real time PCR (qPCR/rtPCR) has been used in numerous fields for the sequence-specific detection and quantification of nucleic acids1,2. Within the relatively new field of eDNA research, use of these assays with a standard curve for quantification of copies of target DNA per volume or weight of eDNA sample has now become routine practice. Mitochondrial DNA sequences are generally targeted in eDNA assays because the mitochondrial genome is present in thousands of copies per cell, but assays for nuclear DNA or RNA sequences are also possible. It is vital to understand that published assays for eDNA samples are not always equal in performance. An assay&#8217;s reliability in detecting only the DNA of a target species (i.e., specificity) and detection of low quantities of target DNA (i.e., sensitivity) may vary considerably due to differences in how the assay was designed, selected, optimized and tested. Reporting quantitative measures of assay performance has been previously largely overlooked, but recently standards to improve transparency in assay development are emerging3,4,5,6,7,8.
Optimization and reporting of assay performance aids in study design and interpretation of eDNA survey results. Assays that cross-react with non-target species DNA could lead to false positive detections, while assays with poor sensitivity may fail to detect the target species DNA even when it is present in the sample (false negatives). An understanding of assay sensitivity and selectivity will help inform the sampling effort needed to detect rare species. Because there are many natural sources of variation in eDNA, studies must limit controllable sources of variation as much as possible, including fully optimizing and characterizing the eDNA assay3.
Conditions that directly affect an assay&#8217;s specificity or sensitivity will change the assay&#8217;s performance. This can occur under different laboratory conditions (i.e., different reagents, users, machines, etc.). Therefore, this protocol should be revisited when applying an assay under new conditions. Even assays well-characterized in the literature should be tested and optimized when adopted by a new laboratory or when using different reagents (e.g., master-mix solution)5,9. Assay specificity may change when applied to a different geographic region, because the assay is being applied to samples from a new biotic community that may include non-target species that the assay has not been tested against, and genetic variation in the target species may occur. Again, the assay should be re-assessed when used in a new location. Field conditions differ from laboratory conditions because in the field PCR inhibitors are more likely to be present in samples. PCR inhibitors directly affect the amplification reaction and thus affect assay performance. For this reason, an internal positive control is required when developing an eDNA assay.
Finally, environmental conditions in the field can affect the target species&#39; DNA molecules and their capture through DNA degradation, transport and retention. Furthermore, different protocols for DNA collection and extraction vary in their efficiency and ability to retain DNA. However, it is important to note that these processes affect the detectability of eDNA but not a molecular assays&#8217; performance. Thus, detectability of DNA from the target species in field samples is a function of both the technical performance of the qPCR assay as well as field conditions and collection, storage, and extraction protocols. When using a well characterized and highly performing assay, users can feel confident in the assay&#8217;s capabilities; allowing researchers to now focus on understanding the external assay factors (i.e., environmental variables, differences in capture or extraction protocols) affecting eDNA detection.
Here we focus specifically on assay technical performance through rigorous design and optimization. We demonstrate the protocol using a probe-based assay developed for the detection of a freshwater mussel, the mucket (Actinonaias ligamentina), from water sampled in the Clinch River, USA. Recently Thalinger et al. (2020) presented guidelines for validation of targeted eDNA assays. Assay design following our protocol will bring an assay to Thalinger et al.&#8217;s level 4 plus an additional step towards level 56. At this point an assay&#8217;s technical performance will be optimized and it will be ready for regular use in laboratory and field applications. Further use of the assay in laboratory, mesocosm, and field experiments can then address questions regarding eDNA detection and factors influencing detectability, the final steps for level 5 validation6.

<table>
	<tbody>
		<tr>
			<td>96 Place Reversible Racks with Covers</td>
			<td>Globe Scientific</td>
			<td>456355AST</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean gloves (ie. latex, nitrile, etc.)</td>
			<td>Kimberly-Clark</td>
			<td>43431, 55090</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX96 Touch Real-Time PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>1855196</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Premium Microcentrifuge Tubes: 1.5mL</td>
			<td>Fisher Scientific</td>
			<td>5408129</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Premium Microcentrifuge Tubes: 2.0mL</td>
			<td>Fisher Scientific</td>
			<td>2681332</td>
			<td></td>
		</tr>
		<tr>
			<td>Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, white/clear</td>
			<td>Bio-Rad</td>
			<td>#HSP9601</td>
			<td></td>
		</tr>
		<tr>
			<td>IPC forward and reverse primers</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td>none</td>
			<td>custom product</td>
		</tr>
		<tr>
			<td>IPC PrimeTime qPCR Probes</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td>none</td>
			<td>custom product</td>
		</tr>
		<tr>
			<td>IPC Ultramer DNA Oligo synthetic template</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td>none</td>
			<td>custom product</td>
		</tr>
		<tr>
			<td>Labnet MPS 1000 Compact Mini Plate Spinner Centrifuge for PCR Plates</td>
			<td>Labnet</td>
			<td>C1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge machine</td>
			<td>Various</td>
			<td>-</td>
			<td>Any microcentrifuge machine that hold 1.5mL and 2.0mL tubes is typically okay.</td>
		</tr>
		<tr>
			<td>Microseal &#39;B&#39; PCR Plate Sealing Film, adhesive, optical</td>
			<td>Bio-Rad</td>
			<td>MSB1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free Water (not DEPC-Treated)</td>
			<td>Invitrogen</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips GP LTS 1000 &#181;L F 768A/8</td>
			<td>Rainin</td>
			<td>30389272</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips GP LTS 20 &#181;L F 960A/10</td>
			<td>Rainin</td>
			<td>30389274</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips GP LTS 200 &#181;L F 960A/10</td>
			<td>Rainin</td>
			<td>30389276</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Rainin</td>
			<td>Various</td>
			<td>Depending on lab preference, manual or electronic pipettes can be used at various maximum volumes.</td>
		</tr>
		<tr>
			<td>TaqMan Environmental Master Mix 2.0</td>
			<td>Thermo Fisher Scientific</td>
			<td>4396838</td>
			<td></td>
		</tr>
		<tr>
			<td>Target forward and reverse primers</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td>none</td>
			<td>custom product</td>
		</tr>
		<tr>
			<td>Target PrimeTime qPCR Probes</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td>none</td>
			<td>custom product</td>
		</tr>
		<tr>
			<td>Target synthetic gBlock gene fragment</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td>none</td>
			<td>custom product. used for qPCR standard dilution series</td>
		</tr>
		<tr>
			<td>TE Buffer</td>
			<td>Invitrogen</td>
			<td>AM9849</td>
			<td></td>
		</tr>
		<tr>
			<td>VORTEX-GENIE 2 VORTEX MIXER</td>
			<td>Fisher Scientific</td>
			<td>50728002</td>
			<td></td>
		</tr>
	</tbody>
</table>

development and testing of species-specific quantitative pcr assays for environmental dna applications

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The use of environmental DNA (eDNA) samples for detecting macrobiota is one such group of methods that is rapidly becoming popular and being implemented in national management programs. Here we focus on the development of species-specific targeted assays for probe-based quantitative PCR (qPCR) applications. Using probe-based qPCR offers greater specificity than is possible with primers alone. Furthermore, the ability to quantify the amount of DNA in a sample can be useful in our understanding of the ecology of eDNA and the interpretation of eDNA detection patterns in the field. Careful consideration is needed in the development and testing of these assays to ensure the sensitivity and specificity of detecting the target species from an environmental sample. In this protocol we will delineate the steps needed to design and test probe-based assays for the detection of a target species; including creation of sequence databases, assay design, assay selection and optimization, testing assay performance, and field validation. Following these steps will help achieve an efficient, sensitive, and specific assay that can be used with confidence. We demonstrate this process with our assay designed for populations of the mucket (Actinonaias ligamentina), a freshwater mussel species found in the Clinch River, USA.

Watch this Scientific Journal Video about Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications at JoVE.com

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

Environmental DNA assays require rigorous design, testing, optimization and validation before the collection of field data can begin. Here, we present a protocol to take users through each step of designing a species-specific, probe-based qPCR assay for the detection and quantification of a target species DNA from environmental samples.

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The ...

Research

JoVE Journal

Environment

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