This work was supported by NSERC Alliance Covid-19 Grant (Award No. 431401363, 2020-2021, Drs. Yuan and Uyaguari-D&#237;az). MUD would like to thank the University Research Grants Program (Award No. 325201). Both JF and JZA are supported by the Visual and Automated Disease Analytics (VADA) graduate training program. KY and JF both received fellowships from the Mitacs Accelerate program. MUD and his laboratory members (KY, JF, JZA) are supported by NSERC-DG (RGPIN-2022-04508) and the Research Manitoba New Investigator Operating grant (No 5385). Special thanks to the City of Winnipeg, Manitoba. This research was conducted at the University of Manitoba. We would like to acknowledge that the University of Manitoba campuses are located on the original lands of Anishinaabeg, Cree, Oji-Cree, Dakota, and Dene peoples and on the homeland of the M&#233;tis Nation.

1. Comparison of serial ultrafiltration and skimmed milk flocculation to concentrate viruses in wastewater samples
<ol>
	<li>Sample preparation
	<ol>
		<li>Collect 2 L of 24 h flow-proportional composite raw (influent) wastewater samples. Samples were collected from the three major wastewater treatment plants (WWTPs) in Winnipeg, Canada, during the summer and fall of 2020 (Table 1).</li>
		<li>Transport the samples to the laboratory in light-proof bottles in an icebox and process them within 24 h. Collect wastewater physico-chemical and biological characteristics data.</li>
	</ol>
	</li>
	<li>Virus concentration assays 
	NOTE: A sequential ultrafiltration method and an organic flocculation method, namely skimmed milk flocculation (SMF), were applied to assess the total recovery efficiency of each method using Armored RNA as a test virus23. Other researchers have also used Armored RNA as a control virus for assessing the recovery of concentration methods for enveloped viruses, such as the family Coronaviridae22,24,25,26.
	<ol>
		<li>For the addition of Armored RNA, set the wastewater test volumes for the ultrafiltration and SMF methods as 140 mL and 500 mL, respectively. Using a pipettor, spike 5 x 104 copies of Armored RNA into six fresh wastewater samples collected on one date and six fresh wastewater samples on another date, for each ultrafiltration method; further, spike six stored (at 4 &#176;C) wastewater samples collected on a third, later date and processed days later for SMF. 
		NOTE: Each of the six sample sets collected on different dates was composed of two samples from each of the three WWTPs. In our study, the first set of fresh wastewater samples were collected on July 8th, 2020, the second on September 30th, 2020, and the wastewater samples to be stored for SMF on October 14th, 2020.</li>
		<li>Stir for 30 min at 4 &#176;C to inoculate and homogenize the Armored RNA in the samples using a magnetic stirrer.</li>
		<li>Remove all particles or cells larger than 0.2 &#956;m and perform ultrafiltration.
		<ol>
			<li>Filter the spiked wastewater samples (120 mL from each 140 mL spiked sample) through cheesecloth and low-protein binding, 0.45 &#181;m and 0.2 &#181;m, 47 mm membrane disc filters, respectively, to remove large particles, sediments, eukaryotes, and bacteria27. Process the filtered samples using the ultrafiltration methods described below. 
			NOTE: The wastewater samples collected on July 8th and September 30th were used for ultrafiltration methods with 3,000 x g and 7,500 x g, respectively.</li>
			<li>For sequential ultrafiltration at 3,000 x g (UF-3k x g), concentrate a total of 120 mL of the test sample collected on the first date at 3,000 x g for 30 min by loading 60 mL of the sample twice to approximately 5 mL using a brand A centrifugal device, 30-kDa. Then, concentrate down the 5 mL volume at 3,000 x g for 30 min using a brand B centrifugal device, 30-kDa, to 500-1,200 &#181;L.</li>
			<li>For sequential ultrafiltration at 7,500 x g (UF-7.5k x g), change the centrifugation speed for the brand B centrifugal device from 3,000 x g to 7,500 x g to obtain smaller final volumes, as per the manufacturer&#39;s recommendations, and keep the centrifugation speed of the brand A centrifugal device the same. 
			NOTE: The tests were run with the samples collected on September 30th.</li>
		</ol>
		</li>
		<li>For SMF, perform the steps outlined below.</li>
		<li>Spike the wastewater samples (500 mL each) to directly concentrate using the SMF protocol16,18, without prefiltration with cheesecloth and low-protein binding membrane disc filters, as described below.</li>
		<li>Dissolve 0.5 g of skimmed milk powder in 50 mL of synthetic seawater to obtain a 1% (w/v) skimmed milk solution and carefully adjust the pH of the solution to 3.5 using 1 N HCl. 
		NOTE: Acidification makes viruses aggregate and precipitate out of water due to the change in their isoelectric point16 and improves the solubility of milk powder28.</li>
		<li>Add 5 mL of skimmed milk solution to 500 mL of raw wastewater samples to obtain a final concentration of skimmed milk of 0.01% (w/v).</li>
		<li>Stir the samples for 8 h at medium speed and allow the formed flocs to settle for another 8 h at room temperature.</li>
		<li>Remove the supernatants carefully using serological pipettes without disturbing the settled flocs. Transfer a final volume of 50 mL containing the flocs to centrifuge tubes and centrifuge at 8,000 x g for 30 min at 8 &#176;C.</li>
		<li>Carefully scrap the pellets using a sterilized spatula, and resuspend the remaining pellets in the tubes in 250 &#181;L of 0.2 M sodium phosphate buffer (pH 7.5) after the supernatant is removed. Transfer the scraped and resuspended pellets to the same 1.5 mL mini-centrifuge tubes.</li>
	</ol>
	</li>
	<li>Perform viral RNA extraction from viral concentrates of wastewater samples and recovery efficiency assays using an RNA extraction kit with a 25:24:1 ratio of phenol:chloroform:isoamyl alcohol and &#946;-mercaptoethanol, according to the manufacturer&#39;s instructions, to improve the extraction efficiency. Finally, elute the RNA in 50 &#181;L of elution buffer.</li>
	<li>Real time-quantitative polymerase chain reaction (RT-qPCR) analysis
	<ol>
		<li>Quantify Armored RNA using tenfold dilutions (7.8 x 104 to 7.8 gene copies) of a synthetic single-stranded DNA or gBlock construct. See Table 2 for the primers and probe sets that detect and quantify the Armored RNA.</li>
		<li>Design primer and probe sets using the primer design tool29 and target a 95 bp region (gBlock construct) within the Armored RNA genome.</li>
		<li>Obtain calibration curves for each RT-qPCR run. Include negative controls in each qPCR run. Run standards, samples, and non-template controls in triplicate.</li>
		<li>Prepare each 10 &#181;L RT-qPCR mixture in the following order: 2.5 &#181;L of 4x master mix, 400 nM of each primer, 200 nM probe, and 2.5 &#181;L of template, as well as ultrapure DNAse/RNAse- free distilled water.</li>
		<li>Perform thermal cycling reactions at 50 &#176;C for 5 min, followed by 45 cycles of 95 &#176;C for 10 s and 60 &#176;C for 30 s on a real-time PCR system. If the CT was &#60;40, consider the sample Armored RNA positive.</li>
		<li>Conduct qPCR and RT-qPCR tests using bovine serum albumin with raw sewage samples to assess the possibility of inhibitors or contaminants such as humic acids24. No significant differences were observed between samples with and without the enzyme.</li>
	</ol>
	</li>
	<li>Calculate the concentrations from the standard curve. Calculate the recovery efficiency percentage by dividing the recovered concentration by the spiked concentration.</li>
	<li>Transform gene copy numbers using the log10 function for analysis. Run a general linear model using a statistical analysis tool on the qPCR data. Apply Tukey&#39;s test to detect differences among the methods and treatments. Take a p-value of 0.05 to be a minimum significance level for the test.</li>
</ol>
2. Filtration and concentration of DNA and RNA viruses for water-based epidemiology
<ol>
	<li>For environmental surface water collection, collect 10 L of environmental surface water samples and use 10 L of deionized water samples for background control. Store all the samples in a 4 &#176;C room until processing within 24 h following sample collection.</li>
	<li>Perform concentration of viruses from environmental samples with SMF using the steps described below.
	<ol>
		<li>Perform capsule and vacuum filtration using&#160;high-capacity groundwater sampling capsules and membrane disc filters of 0.45 &#181;m, 0.2 &#181;m, and 0.1 &#181;m sizes to minimize noise during metagenomic sequencing, from larger fractions such as micro-eukaryotes and bacteria to smaller ones such as viruses.</li>
		<li>Prepare a 1% weight-by-volume pre-flocculated skimmed milk solution in 1.32 L of synthetic seawater with 13.2 g of skimmed milk powder. Adjust the pH of this suspension to 3.5 using 1 N HCl.</li>
	</ol>
	</li>
	<li>Adjust the pH of the environmental surface water samples to 3.5 using 1 N HCl.</li>
	<li>Transfer 100 mL of the pre-flocculated acidified skimmed milk solution to 10 L acidified (pH 3.5) environmental water samples (final skimmed milk concentration of 0.01% [w/v]).</li>
	<li>Using a magnetic stirrer and a magnetic stir bar, stir the samples for 8 h at room temperature and allow the flocs to sediment by gravity for an additional 8 h. Without disturbing the flocs, carefully remove the supernatant using a vacuum pump.</li>
	<li>Aliquot and balance the remaining flocs according to the weight in 50 mL centrifuge tubes and spin them down at 8,000 x g for 30 min at 4 &#176;C.</li>
	<li>Discard the supernatant and dissolve the resulting pellet (which theoretically contains viruses of interest) in 200 &#181;L of 0.2 M sodium phosphate buffer.</li>
	<li>Treat the dissolved pellet with DNase I and RNase A, following the manufacturer&#39;s instructions, to eliminate free DNA and RNA present that may be co-precipitated owing to the kit used for nucleic acid extraction. Inactivate the DNase I and RNase A following the manufacturer&#39;s instructions.</li>
	<li>Extract total nucleic acids from the dissolved pellet using a DNA/RNA extraction kit, as per the manufacturer&#39;s instructions.</li>
	<li>Quantify the total amount of extracted DNA and RNA from effluent samples using a fluorometer. Samples with concentrations &#62;1 ng/&#181;L are considered optimal for DNA quantification and sequencing.</li>
	<li>Perform qPCR analysis to target enteric viruses of interest and high-throughput sequencing to identify the viral community structure.</li>
</ol>
3. Direct precipitation and concentration of microbial fractions for water-based epidemiology
<ol>
	<li>Collect 2 L of water samples from protected and impacted waterways. If the sample contains a considerable amount of debris, use a cheesecloth to remove them. 
	NOTE: Collecting a biological duplicate per sample is highly recommended.</li>
	<li>Prepare a 1% (w/v) skimmed milk solution by dissolving 4.4 g of skimmed milk powder in 440 mL of synthetic seawater. Adjust the pH of this suspension to 3.5 with 1 N HCl.</li>
	<li>Adjust the pH of the freshwater samples to 3.5 using 1 N HCl.</li>
	<li>Add 20 mL of skimmed milk solution to the previously pH-adjusted 2 L freshwater samples (final skimmed milk concentration of 0.01% [w/v]). Stir the samples at room temperature for 8 h.</li>
	<li>Allow the flocs to sediment by gravity for another 8 h. Carefully remove the supernatants with a peristaltic pump. Transfer the sedimented flocs to a 50 mL centrifuge tube and spin them down at 8,000 x g for 30 min.</li>
	<li>Discard the supernatant and resuspend the pellets with 200 &#181;L of 0.2 M sodium phosphate buffer. Store the samples at -20 &#176;C or proceed to select ~ 0.5 g of the floc for microbial DNA extraction by using the nucleic acid isolation kit of choice, following the manufacturer&#39;s instructions.</li>
	<li>Determine the DNA nucleic acid concentration and purity with a high sensitivity fluorometer. Samples with concentrations &#62;1 ng/&#181;L should be optimal for DNA quantification and sequencing.</li>
</ol>

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

One of the critical steps in this study is the elimination of solid particles by applying a prefiltration step with 0.2 &#181;m and 0.45 &#181;m membrane filters. Considering the partitioning of viruses into solid particles, especially enveloped viruses, prefiltration can cause a significant loss in viral recovery30. While a prefiltration step for ultrafiltration methods is almost always necessary for environmental and wastewater samples to prevent ultrafiltration devices from clogging, prefiltrat...

Determining the effective concentration of microorganisms in surface and wastewater samples for microbial community analysis&#160;and&#160;epidemiology studies, is one of the important steps for monitoring and predicting the course of outbreaks in communities1,2. The COVID-19 pandemic, unfolded the importance of improving concentration methods. COVID-19 emerged in late 2019 and, as of March 2023, still poses a threat to human health, social life, and the economy. Effective surveillance and control strategies to alleviate the impacts of COVID-19 outbreaks in communities have become an important research topic, as new waves and variants of COVID-19 have been emerging in addition to the rapid transmission and spread of the virus, as well as unreported and undiagnosed asymptomatic cases3,4,5. The use of wastewater-based epidemiology for COVID-19 by civil society organizations, government agencies, and public or private utilities has been helpful in providing rapid outbreak-related information and mitigating the impacts of COVID-19 outbreaks6,7,8,9. However, the concentration of SARS-CoV-2, an enveloped RNA virus, in wastewater samples still poses challenges10. For example, one of these challenges is the partitioning of SARS-CoV-2 in wastewater solids, which may impact recovery when the solids are eliminated during concentration11. If this is the case, the focus of quantification/assessment should be on both solid and aqueous phases of environmental water samples, rather than the aqueous phase only. Furthermore, the choice of concentration method can be modified based on downstream tests and analyses. The concentration of virus particles and pathogens from environmental samples has become an urgent research topic with developments in sequencing and microbiome fields.
Various virus concentration methods have been applied in the field of virus concentration from environmental water and wastewater samples. Some commonly used methods are filtration, skimmed milk flocculation (SMF), adsorption/elution, and polyethylene glycol precipitation12-17. Among them, SMF has been considered a cheap and effective method, successfully tested, and applied for recovering viruses, including SARS-CoV-2, from wastewater and surface waters12,15,16,18. The SMF procedure is a relatively new approach that has gained increased recognition among many environmental studies as an appropriate methodology to simultaneously recover a broad array of microorganisms such as viruses, bacteria, and protozoans from all types of water samples, namely sludge, raw sewage, wastewater, and effluent samples19. When compared to other known methodologies to recover viruses from environmental samples such as ultrafiltration and glycine-alkaline elution, lyophilization-based approach, or ultracentrifugation and glycine-alkaline elution, SMF has been reported as the most efficient method with higher viral recovery and detection rates18,20. In the present study, we used Armored RNA as a test virus to assess the recovery efficiency of virus concentration methods, including tests for assessing SARS-CoV-2 recovery21,22.
Here, we tested wastewater and environmental water samples to demonstrate the utility of SMF and a sequential ultrafiltration method to concentrate microbial fractions for quantitative polymerase chain reaction (qPCR), sequence-based metagenomics, and deep-amplicon sequencing. SMF is a relatively cheaper method and optimal for a larger volume of samples compared to ultrafiltration methods. The idea of using a sequential ultrafiltration method arose from the necessity to decrease the final volume of the viral concentrates during the COVID-19 pandemic, when the supply of the commonly used ultrafiltration devices was limited, and there was a need for the development of alternative viral concentration methods.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 M sodium phosphate buffer with a pH 7.5</td>
			<td>Alfa Aesar</td>
			<td>J62041AP</td>
			<td>Fisher Scientific, Fair Lawn, NJ, USA</td>
		</tr>
		<tr>
			<td>0.2 &#956;m 47-mm Supor-200 membrane disc filters</td>
			<td>VWR</td>
			<td>66234</td>
			<td>Pall Corporation, Ann Arbor, MI</td>
		</tr>
		<tr>
			<td>0.45 &#956;m 47-mm Supor-200 membrane disc filters</td>
			<td>VWR</td>
			<td>60043</td>
			<td>Pall Corporation, Ann Arbor, MI</td>
		</tr>
		<tr>
			<td>4X TaqMan Fast Virus 1-Step Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4444432</td>
			<td>Life Technologies, Carlsbad, CA, USA</td>
		</tr>
		<tr>
			<td>Armored RNA Quant IPC-1 Processing Control</td>
			<td>Asuragen</td>
			<td>49650</td>
			<td>Asuragen, Austin, TX, USA</td>
		</tr>
		<tr>
			<td>Brand A, Jumbosep Centrifugal Device, 30-kDa</td>
			<td>Pall&#160;</td>
			<td>OD030C65</td>
			<td>Pall Corporation, Ann Arbor, MI</td>
		</tr>
		<tr>
			<td>Brand B, Microsep Advance Centrifugal Device, 30-kDa</td>
			<td>Pall</td>
			<td>MCP010C46</td>
			<td>Pall Corporation, Ann Arbor, MI</td>
		</tr>
		<tr>
			<td>Centrifuge tubes (50 ml)&#160;</td>
			<td>Nalgene</td>
			<td>3119-0050PK</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Invitrogen</td>
			<td>18047019</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>Dyna Mag-2</td>
			<td>Invitrogen</td>
			<td>12027</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>GWV High Capacity Groundwater Sampling Capsules - 0.45 &#181;m</td>
			<td>Pall</td>
			<td>12179</td>
			<td>Pall Corporation, Ann Arbor, MI</td>
		</tr>
		<tr>
			<td>Hydrochloric acid, 1N standard solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>AC124210025</td>
			<td>Fisher Scientific, Fair Lawn, NJ, USA</td>
		</tr>
		<tr>
			<td>MagMAX Microbiome Ultra Nucleic Acid Isolation Kit</td>
			<td>Applied biosystems</td>
			<td>A42358</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>Nuclease free water</td>
			<td>Promega</td>
			<td>P1197</td>
			<td>Promega Corporation, Fitchburg, WI, USA</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Masterflex, Cole-Parmer instrument</td>
			<td>7553-20</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>pH meter&#160;</td>
			<td>Denver instrument</td>
			<td>RK-59503-25</td>
			<td>Cole-Parmer. This product has been discontinued</td>
		</tr>
		<tr>
			<td>Phenol:chloroform:isoamyl alcohol 25:24:1</td>
			<td>Invitrogen</td>
			<td>15593031</td>
			<td>Fisher Scientific, Fair Lawn, NJ, USA</td>
		</tr>
		<tr>
			<td>Primers and probe sets</td>
			<td>IDT</td>
			<td></td>
			<td>Integrated DNA Technologies, Inc., Coralville, IA, USA</td>
		</tr>
		<tr>
			<td>Qiagen All-prep DNA/RNA power microbiome kit</td>
			<td>Qiagen</td>
			<td></td>
			<td>Qiagen Sciences, Inc., Germantown, MD, USA</td>
		</tr>
		<tr>
			<td>QuantStudio 5 Real-Time PCR System</td>
			<td>Thermo Fisher Scientific</td>
			<td>A34322</td>
			<td>Life Technologies, Carlsbad, CA, USA</td>
		</tr>
		<tr>
			<td>Qubit 1X dsDNA High Sensitivity (HS) assay kit</td>
			<td>Invitrogen</td>
			<td>Q33231</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>Qubit 4 Fluorometer, with WiFi</td>
			<td>Invitrogen</td>
			<td>Q33238</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>Qubit RNA High Sensitivity (HS) assay kit</td>
			<td>Invitrogen</td>
			<td>Q32855</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>RNAse A</td>
			<td>Invitrogen</td>
			<td>EN0531</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>RNeasy PowerMicrobiome Kit</td>
			<td>Qiagen</td>
			<td>26000-50</td>
			<td>Qiagen Sciences, Inc., Germantown, MD, USA</td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Difco (BD Life Sciences)</td>
			<td>DF0032173</td>
			<td>Fisher Scientific, Fair Lawn, NJ, USA</td>
		</tr>
		<tr>
			<td>Sodium phosphate buffer</td>
			<td>Alfa Aesar</td>
			<td></td>
			<td>Alfa Aesar, Ottawa, ON, Canada</td>
		</tr>
		<tr>
			<td>Synthetic seawater</td>
			<td>VWR&#160;</td>
			<td>RC8363-1</td>
			<td>RICCA chemical company</td>
		</tr>
		<tr>
			<td>Synthetic single-stranded DNA gBlock</td>
			<td>IDT</td>
			<td></td>
			<td>Integrated DNA Technologies, Inc., Coralville, IA, USA</td>
		</tr>
		<tr>
			<td>VacuCap 90 Vacuum Filtration Devices - 0.1 &#181;m, 90 mm, gamma-irradiated</td>
			<td>Pall</td>
			<td>4621</td>
			<td>Pall Corporation, Ann Arbor, MI</td>
		</tr>
		<tr>
			<td>VacuCap 90 Vacuum Filtration Devices - 0.2 &#181;m, 90 mm, gamma-irradiated</td>
			<td>Pall</td>
			<td>4622</td>
			<td>Pall Corporation, Ann Arbor, MI</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>Fisher Scientific, Fair Lawn, NJ, USA</td>
		</tr>
	</tbody>
</table>

concentration of virus particles from environmental water and wastewater samples using skimmed milk flocculation and ultrafiltration

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The recovery of microbial fractions, including viruses, bacteria, and microeukaryotes from wastewater and environmental water samples&#160;is one of the challenging steps in these approaches. In this study, we focused on the recovery efficiency of sequential ultrafiltration and skimmed milk flocculation (SMF) methods using Armored RNA as a test virus, which is also used as a control by some other studies. Prefiltration with 0.45 &#181;m and 0.2 &#181;m membrane disc filters were applied to eliminate solid particles before ultrafiltration to prevent the clogging of ultrafiltration devices. Test samples, processed with the sequential ultrafiltration method, were centrifuged at two different speeds. An increased speed resulted in lower recovery and positivity rates of Armored RNA.&#160;On the other hand, SMF resulted in relatively consistent recovery and positivity rates of Armored RNA. Additional tests conducted with environmental water samples demonstrated the utility of SMF to concentrate other microbial fractions. The partitioning of viruses into solid particles might have an impact on the overall recovery rates, considering the prefiltration step applied before the ultrafiltration of wastewater samples. SMF with prefiltration performed better when applied to environmental water samples&#160;due to lower solid concentrations in the samples and thus lower partitioning rates to solids. In the present study, the idea of using a sequential ultrafiltration method arose from the necessity to decrease the final volume of the viral concentrates during the COVID-19 pandemic, when the supply of the commonly used ultrafiltration devices was limited, and there was a need for the development of alternative viral concentration methods.

Evaluation of viral RNA concentration methods 
All six samples processed with UF-3k x g were positive and resulted in a 13.38% &#177; 8.14% recovery (Figure 1). Only one sample was positive when the samples were processed with UF-7.5k x g. All samples processed with SMF were positive and resulted in a 15.27% &#177; 2.65% recovery (Figure 1). The average recovery rates of UF-3K x g and SMF were significantly and co...

Watch this Scientific Journal Video about Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration at JoVE.com

Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration

Virus concentration from environmental water and wastewater samples is a challenging task, carried out primarily for the identification and quantification of viruses. While several virus concentration methods have been developed and tested, we demonstrate here the effectiveness of ultrafiltration and skimmed milk flocculation for RNA viruses with different sample types.

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The ...

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1.3K Views.  University of Manitoba. Virus concentration from environmental water and wastewater samples is a challenging task, carried out primarily for the identification and quantification of viruses. While several virus concentration methods have been developed and tested, we demonstrate here the effectiveness of ultrafiltration and skimmed milk flocculation for RNA viruses with different sample types. 

Video: Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most experiments examine only two or a few levels of CA concentration and a single soil type, but if CA can be varied as a gradient from subambient to superambient concentrations on multiple soils, we can discern whether past ecosystem responses may continue linearly in the future and whether responses may vary across the landscape. The Lysimeter Carbon Dioxide Gradient Facility applies a 250 to 500 &#181;l L-1 CA gradient to Blackland prairie plant communities established on lysimeters containing clay, silty clay, and sandy soils. The gradient is created as photosynthesis by vegetation enclosed in in temperature-controlled chambers progressively depletes carbon dioxide from air flowing directionally through the chambers. Maintaining proper air flow rate, adequate photosynthetic capacity, and temperature control are critical to overcome the main limitations of the system, which are declining photosynthetic rates and increased water stress during summer. The facility is an economical alternative to other techniques of CA enrichment, successfully discerns the shape of ecosystem responses to subambient to superambient CA enrichment, and can be adapted to test for interactions of carbon dioxide with other greenhouse gases such as methane or ozone.

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

The Lysimeter Carbon Dioxide Gradient Facility creates a 250 to 500 &#181;l L-1 linear carbon dioxide gradient in temperature-controlled chambers housing grassland plant communities on clay, silty clay, and sandy soil monoliths. The facility is used to determine how past and future carbon dioxide levels affect grassland carbon cycling.

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most ...

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 ...

Comparison of Scale in a Photosynthetic Reactor System for Algal Remediation of Wastewater

An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. In this study, ammonia removal, nitrogen removal and algal growth are compared over an 8-week period in paired sets of small (100 L) and large (1,000 L) reactors designed for algal remediation of landfill wastewater. Contents of the small and large scale reactors were mixed before the beginning of each weekly testing interval to maintain equivalent initial conditions across the two scales. System characteristics, including surface area to volume ratio, retention time, biomass density, and wastewater feed concentrations, can be adjusted to better equalize conditions occurring at both scales. During the short 8-week representative time period, starting ammonia and total nitrogen concentrations ranged from 3.1-14 mg NH3-N/L, and 8.1-20.1 mg N/L, respectively. The performance of the treatment system was evaluated based on its ability to remove ammonia and total nitrogen and to produce algal biomass. Mean &#177; standard deviation of ammonia removal, total nitrogen removal and biomass growth rates were 0.95&#177;0.3 mg NH3-N/L/day, 0.89&#177;0.3 mg N/L/day, and 0.02&#177;0.03 g biomass/L/day, respectively. All vessels showed a positive relationship between the initial ammonia concentration and ammonia removal rate (R2=0.76). Comparison of process efficiencies and production values measured in reactors of different scale may be useful in determining if lab-scale experimental data is appropriate for prediction of commercial-scale production values.

An experimental methodology is presented to compare the performance of small (100 L) and large (1,000 L) scale reactors designed for algae remediation of landfill wastewater. System characteristics, including surface area to volume ratio, retention time, biomass density, and wastewater feed concentrations, can be adjusted based on application.

An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. In this study, ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...