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., Griffith, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+amplification+and+detection+of+specific+DNA+sequences.">Simultaneous amplification and detection of specific DNA sequences.</a> Biotechnology. 10, 5 (1992).</li><li>Mauvisseau, Q., 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=Influence+of+accuracy,+repeatability+and+detection+probability+in+the+reliability+of+species-specific+eDNA+based+approaches.">Influence of accuracy, repeatability and detection probability in the reliability of species-specific eDNA based approaches.</a> Scientific Reports. 9 (1), 580 (2019).</li><li>Hernandez, C., 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=60+specific+eDNA+qPCR+assays+to+detect+invasive,+threatened,+and+exploited+freshwater+vertebrates+and+invertebrates+in+Eastern+Canada.">60 specific eDNA qPCR assays to detect invasive, threatened, and exploited freshwater vertebrates and invertebrates in Eastern Canada.</a> Environmental DNA. , (2020).</li><li>Klymus, K. 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=Reporting+the+limits+of+detection+and+quantification+for+environmental+DNA+assays.">Reporting the limits of detection and quantification for environmental DNA assays.</a> Environmental DNA. , (2019).</li><li>Thalinger, B., 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=A+validation+scale+to+determine+the+readiness+of+environmental+DNA+assays+for+routine+species+monitoring.">A validation scale to determine the readiness of environmental DNA assays for routine species monitoring.</a> bioRxiv. , (2020).</li><li>Helbing, C. 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M., 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=Robust+detection+of+rare+species+using+environmental+DNA:+the+importance+of+primer+specificity.">Robust detection of rare species using environmental DNA: the importance of primer specificity.</a> PLoS One. 8 (3), 59520 (2013).</li><li>Prediger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+design+primers+and+probes+for+PCR+and+qPCR.">How to design primers and probes for PCR and qPCR.</a> IDT. , (2020).</li><li>Thornton, B., Basu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+(qPCR)+primer+design+using+free+online+software.">Real-time PCR (qPCR) primer design using free online software.</a> Biochemistry and Molecular Biology Education. 39, 145-154 (2011).</li><li>Owczarzy, R., 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=IDT+SciTools:+a+suite+for+analysis+and+design+of+nucleic+acid+oligomers.">IDT SciTools: a suite for analysis and design of nucleic acid oligomers.</a> Nucleic Acids Research. 36, 163-169 (2008).</li><li>Kibbe, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OligoCalc:+an+online+oligonucleotide+properties+calculator.">OligoCalc: an online oligonucleotide properties calculator.</a> Nucleic Acids Research. 35, 43-46 (2007).</li><li>Taylor, S. C., 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=The+Ultimate+qPCR+Experiment:+Producing+Publication+Quality,+Reproducible+Data+the+First+Time.">The Ultimate qPCR Experiment: Producing Publication Quality, Reproducible Data the First Time.</a> Trends in Biotechnology. 37 (7), 761-774 (2019).</li><li>Ye, J., 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=Primer-BLAST:+A+tool+to+design+target-specific+primers+for+polymerase+chain+reaction.">Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction.</a> BMC Bioinformatics. 13 (134), 11 (2012).</li><li>Altschul, S. F., Gish, W., Miller, W., Myers, E. W., Lipman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Local+Alignment+Search+Tool.">Basic Local Alignment Search Tool.</a> Journal of Molecular Biology. 215, 403-410 (1990).</li><li>Bustin, S. 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Vol. 0708-V2. , (2020).</li><li>Bustin, S., Huggett, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=qPCR+primer+design+revisited.">qPCR primer design revisited.</a> Biomolecular Detection and Quantification. 14, 19-28 (2017).</li><li>Hoorfar, J., 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=Practical+considerations+in+design+of+internal+amplification+controls+for+diagnostic+PCR+assays.">Practical considerations in design of internal amplification controls for diagnostic PCR assays.</a> Journal of Clinical Microbiology. 42 (5), 1863-1868 (2004).</li><li>Goldberg, C. 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. 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=Successful+molecular+detection+studies+require+clear+communication+among+diverse+research+partners.">Successful molecular detection studies require clear communication among diverse research partners.</a> Frontiers in Ecology and the Environment. 18 (1), 43-51 (2019).</li><li>Kwonga, S., Srivathsana, A., Meier, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+on+DNA+barcoding:+low+species+coverage+and+numerous+unidentified+sequences.">An update on DNA barcoding: low species coverage and numerous unidentified sequences.</a> Cladistics. 28, 6 (2012).</li><li>Rees, H. C., 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=REVIEW:+The+detection+of+aquatic+animal+species+using+environmental+DNA+-+a+review+of+eDNA+as+a+survey+tool+in+ecology.">REVIEW: The detection of aquatic animal species using environmental DNA - a review of eDNA as a survey tool in ecology.</a> Journal of Applied Ecology. 51 (5), 1450-1459 (2014).</li><li>Evans, N. T., Lamberti, G. 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>
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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 ...

development-testing-species-specific-quantitative-pcr-assays-for

Research

JoVE Journal

Environment

12.6K Views.  U.S. Geological Survey. 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.

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

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either "Contamination Detected" or "No Contamination Detected" within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be brought back to the beehive after the bees have foraged on flowers that have been sprayed with pesticides. Pesticide contaminated food can be exchanged between workers which then feed larvae and therefore can potentially affect the development of honey bees. Thus, residual pesticides in the environment can become a chronic damaging factor to honey bee populations and gradually lead to colony collapse. In the presented protocol, honey bee feeding methods are described and applied to either an individual honey bee or to a colony. Here, the insect growth regulator (IGR) pyriproxyfen (PPN), which is widely used to control pest insects and is harmful to the development of honey bee larvae and pupae, is used as the pesticide. The presenting procedure can be applied to other potentially harmful chemicals or honeybee pathogens for further studies.

Herein we present a method to feed pesticide contaminated food to both an individual honey bee and a beehive colony. The procedure evaluates the pesticide effect on individual honey bees by in vivo feeding of basic larval diet and also on the natural condition of beehive colony.

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be ...

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity.

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

Resurrection of Dormant Daphnia magna: Protocol and Applications

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical data that may be used in predictive modelling to forecast evolutionary responses of natural ecosystems to future environmental changes. However, excluding a few exceptional cases, long-term studies are scarce because of logistic difficulties associated with accessing temporal samples. Temporal dynamics are frequently studied in the laboratory or in controlled mesocosm experiments with exceptional studies that reconstruct the evolution of natural populations in the wild.
Here, a standard operating procedure (SOP) is provided to revive or resurrect dormant Daphnia magna, a widespread zooplankton keystone species in aquatic ecosystems, to dramatically advance the state-of-the-art longitudinal data collection in natural systems. The field of Resurrection Ecology was defined in 1999 by Kerfoot and co-workers, even though the first attempts at hatching diapausing zooplankton eggs date back to the late 1980s. Since Kerfoot&#39;s seminal paper, the methodology of resurrecting zooplankton species has been increasingly frequently applied, though propagated among laboratories only via direct knowledge transfer. Here, an SOP is described that provides a step-by-step protocol on the practice of resurrecting dormant Daphnia magna eggs.
Two key studies are provided in which the fitness response of resurrected Daphnia magna populations to warming is measured, capitalizing on the ability to study historical and modern populations in the same settings. Finally, the application of next generation sequencing technologies to revived or still dormant stages is discussed. These technologies provide unprecedented power in dissecting the processes and mechanisms of evolution if applied to populations that have experienced changes in selection pressure over time.

Resurrection of Dormant Daphnia magna: Protocol and Applications

Long-term studies are essential to understanding the process of evolution and the mechanisms of adaptation. Generally, these studies require commitments beyond the life-time of researchers. Here, a powerful method is described that dramatically advances state-of-the-art data collection to generate longitudinal data in natural systems.

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical ...

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species.
This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

In this work, we describe an acute, chronic and multigenerational bioassay to study the effects of single and combined stressors on the Turquoise killifish Nothobranchius furzeri. This protocol is designed to study life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol for performing in situ juvenile exposure tests within oligotrophic river catchments over one-month and three-month periods. Two methods (in both modifications) are presented to evaluate the juvenile growth and survival rate. The methods and modifications differ in value for the locality bioindication and each has its benefits as well as limitations. The sandy cage method works with a large set of individuals, but only some of the individuals are measured and the results are evaluated in bulk. In the mesh cage method, the individuals are kept and measured separately, but a low individual number is evaluated. The open water exposure modification is relatively easy to apply; it shows the juvenile growth potential of sites and can also be effective for water toxicity testing. The within-bed exposure modification needs a high workload but is closer to the conditions of a natural juvenile environment and it is better for reporting the real suitability of localities. On the other hand, more replications are needed in this modification due to its high-hyporheic environment variability.

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

In situ bioindications enable determination of the suitability of an environment for endangered mussel species. We describe two methods based on the juvenile exposure of freshwater pearl mussels in cages to oligotrophic river habitats. Both methods are implemented in variants for open water and hyporheic water environments.

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...