This work was completed with funding from the U.S. Geological Survey.
Any use of trade, firm, or products names is for descriptive purposes only and does not imply endorsement by the U.S. Government.

1. Decontamination of equipment and flocculation reagent preparation
<ol>
	<li>Thoroughly wash all bottles, tubing, and other previously used equipment with dishwashing detergent and hot water.</li>
	<li>Spray all bottles, tubing, and other previously used equipment with 6% citric acid solution to remove mineral deposits, let stand 20 minutes, and rinse with tap water.</li>
	<li>Add a solution of 10-fold diluted household bleach (0.5-0.6% final concentration of sodium hypochlorite) to all bottles (approximately 10% of ...

<ol>
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F., Miaud, C., Pompanon, F., Taberlet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+detection+using+environmental+DNA+from+water+samples.">Species detection using environmental DNA from water samples.</a> Biology Letters. 4 (4), 423-425 (2008).</li><li>Djurhuus, 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=Evaluation+of+Filtration+and+DNA+Extraction+Methods+for+Environmental+DNA+Biodiversity+Assessments+across+Multiple+Trophic+Levels.">Evaluation of Filtration and DNA Extraction Methods for Environmental DNA Biodiversity Assessments across Multiple Trophic Levels.</a> Frontiers in Marine Science. 4 (314), (2017).</li><li>Hinlo, R., Gleeson, D., Lintermans, M., Furlan, E. <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+maximise+recovery+of+environmental+DNA+from+water+samples.">Methods to maximise recovery of environmental DNA from water samples.</a> PLOS One. 12 (6), 0179251 (2017).</li><li>Majaneva, 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=Environmental+DNA+filtration+techniques+affect+recovered+biodiversity.">Environmental DNA filtration techniques affect recovered biodiversity.</a> Scientific reports. 8 (1), 4682 (2018).</li><li>Aguado, D., 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+Point-of-Use+Method+for+the+Detection+of+Viruses+in+Water+Samples.">A Point-of-Use Method for the Detection of Viruses in Water Samples.</a> Journal of Visualized Experiments. (147), e58463 (2019).</li><li>Bofill-Mas, 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=Cost-effective+Method+for+Microbial+Source+Tracking+Using+Specific+Human+and+Animal+Viruses.">Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses.</a> Journal of Visualized Experiments. (58), e2820 (2011).</li><li>Gonzales-Gustavson, 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=Characterization+of+the+efficiency+and+uncertainty+of+skimmed+milk+flocculation+for+the+simultaneous+concentration+and+quantification+of+water-borne+viruses,+bacteria+and+protozoa.">Characterization of the efficiency and uncertainty of skimmed milk flocculation for the simultaneous concentration and quantification of water-borne viruses, bacteria and protozoa.</a> Journal of Microbiological Methods. 134, 46-53 (2017).</li><li>Payment, P., Fortin, S., Trudel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferric+chloride+flocculation+for+nonflocculating+beef+extract+preparations.">Ferric chloride flocculation for nonflocculating beef extract preparations.</a> Applied Environmental Microbiology. 47 (3), 591-592 (1984).</li><li>Poulos, B. T., John, S. G., Sullivan, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+Chloride+Flocculation+of+Bacteriophages+from+Seawater.">Iron Chloride Flocculation of Bacteriophages from Seawater.</a> Methods Molecular Biology. 1681, 49-57 (2018).</li><li>Zhang, Y., Riley, L. K., Lin, M., Hu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lanthanum-based+concentration+and+microrespirometric+detection+of+microbes+in+water.">Lanthanum-based concentration and microrespirometric detection of microbes in water.</a> Water Research. 44 (11), 3385-3392 (2010).</li><li>Zhang, Y., Riley, L. K., Lin, M., Hu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+low-density+Escherichia+coli+and+Helicobacter+pylori+suspensions+in+water.">Determination of low-density Escherichia coli and Helicobacter pylori suspensions in water.</a> Water Research. 46 (7), 2140-2148 (2012).</li><li>Schill, W. B., Galbraith, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+the+undetectable:+Characterization,+optimization,+and+validation+of+an+eDNA+detection+assay+for+the+federally+endangered+dwarf+wedgemussel,+Alasmidonta+heterodon+(Bivalvia:+Unionoida).">Detecting the undetectable: Characterization, optimization, and validation of an eDNA detection assay for the federally endangered dwarf wedgemussel, Alasmidonta heterodon (Bivalvia: Unionoida).</a> Aquatic Conservation: Marine and Freshwater Ecosystems. 29 (4), 603-611 (2019).</li><li>Zhang, Y., Riley, L. K., Lin, M., Purdy, G. 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Although this technique is straightforward, some steps are critical to success. The collection and transfer of the flocs must be done completely so as not to lose DNA. Residual material must be gathered using strategic rinses. The described protocol uses a commercial DNA extraction kit for the final processing of flocculated material. Other extraction methods may perform similarly or may be superior in performance, but modifications of this part of the protocol have not been exhaustively tested.
<p class="jove_conten...

Before the mid-2000s, the term &#8220;environmental DNA&#8221; was generally used to refer to DNA obtained from water and soil microbes although some use for the detection of human and animal DNA for purposes of fecal source tracking or non-native species detection had begun1,2,3. Presently, environmental DNA is generally used to describe DNA from cells sloughed from eukaryotic organisms, and the analysis of this DNA has become a widely used management tool. The detection of cryptic species including those that are invasive or species at risk is the goal, typically accomplished by testing water or sediment for the presence of the characteristic DNA signatures of the target species. Studies may focus on a single species of interest using a specific PCR assay (active sampling), or many species may be detected simultaneously using metabarcoding and massively parallel sequencing.
Many improvements in sample collection, sample processing, and data analysis associated with eDNA research have been made during the last two decades. Early studies employed alcohol precipitation to capture DNA from water samples3. While tried and true, the need to analyze large water volumes to increase assay sensitivity make this method unattractive due to the typical requirement for two volumes of ethanol for each volume of water sample.
Later studies have generally used membrane filtration, rationalizing that eDNA is present in a continuum of states from cellular-bound to freely dissolved, but that dissolved DNA is subject to rapid degradation and therefore that cellular-bound DNA represents the most significant fraction. Filter material and pore size optimization is widely discussed and debated as is the optimal method(s) for extraction of captured eDNA from the filter4,5,6.
A third approach to eDNA capture is flocculation, a process that has been used for centuries to clarify liquids including, water, beer, and wine. Several studies have used flocculation to capture virus from natural or potable water sources7,8,9,10,11. These studies have typically used pre-flocculated skim milk or iron chloride as capture agents. Iron chloride methods typically result in downstream analysis molecular problems due to PCR reaction inhibition12,13. Methods using pre-flocculated skim milk require the advance preparation of the flocculant, pH control, and thus far have only been applied to the capture of virus7,8,9.
Recently, flocculation capture of bacteria and virus based on the use of lanthanum (III) chloride has been studied12,13,14,15. The authors demonstrated that viable pathogens could be collected by this method as the binding of lanthanum chloride was effective at low concentration (0.2 mM). Furthermore, lanthanum flocculation is reversible via chelation under mild conditions in contrast to that of iron chloride.
Thus, flocculation using lanthanum chloride to capture biological particulates is attractive given that only a small amount of added concentrated flocculating agent is required, no strict pH control is needed, and sample volumes are scalable from milliliters to gallons. As applied to the capture of eDNA, cellular, sub-cellular (mitochondria and chloroplasts) as well as dissolved DNA are readily recovered. Bacteria and virus are collected simultaneously allowing for pathogen testing and (or) additional ecological studies from a single water sample. A flocculation protocol using lanthanum chloride is presented that enables collection of dissolved and particulate-bound nucleic acids from water samples.

<table>
	<tbody>
		<tr>
			<td>0.5M EDTA, pH 8.0</td>
			<td>Available from several commercial sources</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 x TE Buffer, pH 8.0</td>
			<td>Available from several commercial sources</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Conical Centrifuge Tubes</td>
			<td>Single use</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>16 oz. PET Clear Square Bottles</td>
			<td>Reusable with cleaning and decontamination</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>38/400 Polypropylene Cap with Pressure Sensitive Liner</td>
			<td>Leave caps loosely attached until use. Cap tightly to seal pressure sensitive liner.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical Centrifuge Tubes</td>
			<td>Single use</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6% Citric Acid Solution</td>
			<td>Use to remove mineral deposits from reusable sample bottles.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fecal/Soil Microbe Kit</td>
			<td>Use inhibitor removal resin or column</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lanthanum (III) chloride heptahydrate</td>
			<td>100 mM Stock Solution</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic DC Pump</td>
			<td>Any peristaltic pump will do, or can be syphoned</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyacryl Carrier</td>
			<td>10% linear polyacrylamide</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>1 M, filter sterilized and stored at room temperaruture</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

capture of environmental dna (edna) from water samples by flocculation

The analysis of environmental DNA (eDNA) has become a widely used approach to problem solving in species management. The detection of cryptic species including invasive and (or) species at risk is the goal, typically accomplished by testing water and sediment for the presence of characteristic DNA signatures. Reliable and efficient procedures for the capture of eDNA are required, especially those that can be performed easily in the field by personnel with limited training and citizen scientists. The capture of eDNA using membrane filtration is widely used currently. This approach has inherent issues that include the choice of filter material and porosity, filter fouling, and time required on site for the process to be performed. Flocculation offers an alternative that can be easily implemented and applied to sampling regimes that strive to cover broad territories in limited time.

Eight 500 mL water samples (450 mL each) taken from an aquarium housing two Eastern painted turtles (Chrysemys picta picta) were used to prepare eDNA using the described protocol. Another eight 500 mL water samples were filtered over 47 mm glass fiber filters of 1.5 &#181;m porosity containing no binder and eDNA was extracted using the same extraction method as used for the flocculation protocol. DNA prepared using both methods was analyzed using quantitative PCR that targets the mitochondrial control region of ...

Read this Scientific Article Protocol about Capture of Environmental DNA eDNA from Water Samples by Flocculation at JoVE.com

Capture of Environmental DNA eDNA from Water Samples by Flocculation

Environmental DNA (eDNA) is typically captured from water samples using alcohol precipitation, filtration, or flocculation. Presented here is an alternative protocol that uses lanthanum chloride to enable collection of dissolved and particulate-bound nucleic acids from water samples containing eukaryotic cells, prokaryotic cells, and virus.

Capture of Environmental DNA (eDNA) from Water Samples by Flocculation

The analysis of environmental DNA (eDNA) has become a widely used approach to problem solving in species management. The detection of cryptic species ...