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 ...

<ol>
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J., Cocks, C., Green, J. 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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=Making+waves:+Wastewater+surveillance+of+SARS-CoV-2+for+population-based+health+management.">Making waves: Wastewater surveillance of SARS-CoV-2 for population-based health management.</a> Water Research. 184, 116181 (2020).</li><li>Wu, F., 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=SARS-CoV-2+RNA+concentrations+in+wastewater+foreshadow+dynamics+and+clinical+presentation+of+new+COVID-19+cases.">SARS-CoV-2 RNA concentrations in wastewater foreshadow dynamics and clinical presentation of new COVID-19 cases.</a> The Science of the Total Environment. 805, 150121 (2022).</li><li>Kantor, R. S., Nelson, K. L., Greenwald, H. D., Kennedy, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+measuring+the+recovery+of+SARS-CoV-2+from+wastewater.">Challenges in measuring the recovery of SARS-CoV-2 from wastewater.</a> Environmental Science &amp; Technology. 55 (6), 3514-3519 (2021).</li><li>Chik, A. H. 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=Comparison+of+approaches+to+quantify+SARS-CoV-2+in+wastewater+using+RT-qPCR:+Results+and+implications+from+a+collaborative+inter-laboratory+study+in+Canada.">Comparison of approaches to quantify SARS-CoV-2 in wastewater using RT-qPCR: Results and implications from a collaborative inter-laboratory study in Canada.</a> Journal of Environmental Sciences. 107, 218-229 (2021).</li><li>Hjelms&#248;, M. 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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 ...

Water-based and wastewater epidemiology has emerged as an alternative method to monitor and predict the course of outbreaks in communities. The skimmed milk flocculation method represents an alternative approach to concentrate microbes. Skimmed milk flocculation method represents a versatile, cheap, and easy hands-off method to concentrate microbial fractions with no significant differences compared to ultra filtration or tangential flow filtration approaches.This approach is used for metagenomics or targeted PCR studies. Once the microbial fractions are concentrated, nucleic acid extraction can be conducted using commercial extraction kits or in-house protocol. The skimmed milk flocculation method is relatively easier for large or small volumes of water or wastewater samples.A background control with Milli-Q water should be included as part of this method. Demonstrating the procedure will be Kadir Yanac, Jhannelle Francis, and Jocelyn Zambrano-Alvarado, graduate students from Miguel Uyaguari's laboratory. To begin, collect two liters of 24-hour flow proportional composite raw wastewater samples.Transport the samples to the laboratory in light-proof bottles in an icebox and process them within 24 hours. Get the wastewater characteristics data from wastewater treatment plant technical staff. For each ultra filtration method, spike five times 10 to the fourth copies of armored RNA into 140 milliliters of each of six fresh wastewater samples collected on one date and six fresh wastewater samples on another date.Homogenize the armored RNA in the sample by stirring for 30 minutes at four degrees Celsius on a magnetic stir. For skimmed milk flocculation or SMF, prepare 1%skimmed milk solution by dissolving 0.5 grams of skimmed milk powder in 50 milliliters of synthetic seawater. Adjust the pH of SMF to 3.5 using one normal hydrochloric acid.Add five milliliters of skimmed milk solution to 500 milliliters of raw wastewater sample. Stir the sample for eight hours and allow the formed flocks to settle for another eight hours at room temperature. Remove the supernatant using a serological pipette without disturbing the settled flocks.Transfer a final volume of 50 milliliters containing the flocks to a centrifuge tube and centrifuge at 8, 000 G for 30 minutes at four degrees Celsius. Remove the supernatant and carefully scrap the pellet using a sterilized spatula. Resuspend the remaining pellet in 250 microliters of 0.2 molar sodium phosphate buffer.Transfer the scraped and resuspended pellets to the same 1.5 milliliter mini centrifuge tube. Perform viral RNA extraction from viral concentrates of wastewater samples and recovery efficiency assays using an RNA extraction kit with a 25:24:2:1 ratio of phenol to chloroform to isoamyl alcohol and beta mercaptoethanol according to the manufacturer's instructions. Finally, elute the RNA in 50 microliters of elution buffer.For RT-qPCR analysis, quantify armored RNA using tenfold dilutions of a synthetic single stranded DNA or gBlock construct. Obtain calibration curves for each RT-qPCR run. Include negative controls and run the samples in triplicate.Collect 10 liters of environmental surface water sample. Include 10 liters of deionized water for background control. Perform capsule and vacuum filtration using membrane disc filters of 0.45, 0.2, and 0.1 micron sizes to minimize noise during metagenomics sequencing.After filtration, adjust the pH of the water sample to 3.5 using one normal hydrochloric acid. Prepare a 1%pre-flocculated skimmed milk solution and adjust the pH to 3.5. Add 100 milliliters of the acidified skimmed milk solution to 10 liters of acidified environmental water sample.Stir the sample for eight hours at room temperature on a magnetic shaker and allow the flocks to sediment by gravity for an additional eight hours. After settling, carefully remove the supernatant using a vacuum pump without disturbing the flocks. Aliquot and balance the remaining flocks according to the weight in a 50 milliliter centrifuge tube and centrifuge.Remove the supernatant and dissolve the pellet in 200 microliters of 0.2 molar sodium phosphate buffer. Treat the dissolved pellets with DNase I and RNase A following the manufacturer's instructions to eliminate free DNA and RNA coprecipitated during the extraction process. After incubation, inactivate DNase I and RNase A.Extract nucleic acids from the dissolved pellet using a DNA or RNA extraction kit.Quantify the total amount of extracted DNA or RNA from the environmental surface water sample using a fluorometer. Perform qPCR analysis to target enteric viruses of interest and Illumina sequencing to identify the viral community structure. Collect two liters of water samples from protected and impacted waterways.If the sample contains considerable debris, use a cheesecloth to remove them. Add 20 milliliters of 1%skimmed milk solution to the previously pH adjusted two liter freshwater sample. Stir the sample for eight hours at room temperature and allow the flocks to sediment by gravity for an additional eight hours.After settling, carefully remove the supernatant using a peristaltic pump. Transfer the sedimented flocks to a 50 milliliter centrifuge tube and centrifuge them at 8, 000 G for 30 minutes at four degrees Celsius. Discard the supernatant and resuspend the pellets in 200 microliters of 0.2 molar sodium phosphate buffer.Select around 0.5 grams of the flock for DNA extraction using the nucleic acid isolation kit of choice following the manufacturer's instructions. Determine the DNA concentration and purity using a fluorometer. All six samples processed with ultra filtration at 3, 000 G were positive.In contrast, only one sample was positive when the samples were processed with ultra filtration at 7, 500 G.All samples processed with SMF were positive and resulted in better recovery. The average recovery rates of 3, 000 G and SMF were significantly and consistently higher than ultra filtration at 7, 500 G.Water quality parameters in influenced samples and nucleic acids extracted from environmental water samples collected within Winnipeg across three seasons are summarized here. Samples collected from April to October 2022 from Assiniboine River water yielded the highest DNA concentration from Forested 1 and the lowest concentration was recovered from agricultural site three.It is very important to use the right concentration of skimmed milk and acidify the sample prior to use. This technique can provide a quick screening of different microbial pathogens present in environmental samples such as water and wastewater. Additional modifications can be implemented using solid matrices such as soil or sediments.

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Environment

1.2K Views.  University of Manitoba. Water-based and wastewater epidemiology has emerged as an alternative method to monitor and predict the course of outbreaks in communities. The skimmed milk flocculation method represents an alternative approach to concentrate microbes. Skimmed milk flocculation method represents a versatile, cheap, and easy hands-off method to concentrate microbial fractions with no significant differences compared to ultra filtration or tangential flow filtration approaches.This approach is used for ...