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

Kadir Yana&#231;; Jhannelle Francis; Jocelyn Zambrano-Alvarado; Qiuyan Yuan; Miguel Uyaguari-D&#237;az

doi:10.3791/65058

JoVE Journal > Environment

Ambiente

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

Published: March 17, 2023

doi:

10.3791/65058

Kadir Yanaç¹, Jhannelle Francis², Jocelyn Zambrano-Alvarado², Qiuyan Yuan¹, Miguel Uyaguari-Díaz²

¹Department of Civil Engineering, Price Faculty of Engineering,University of Manitoba, ²Department of Microbiology, Faculty of Science,University of Manitoba

Summary

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.

Abstract

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities. The recovery of microbial fractions, including viruses, bacteria, and microeukaryotes from wastewater and environmental water samples 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 µm and 0.2 µm 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. 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 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.

Introduction

Determining the effective concentration of microorganisms in surface and wastewater samples for microbial community analysis and 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.

Protocol

1. Comparison of serial ultrafiltration and skimmed milk flocculation to concentrate viruses in wastewater samples

Sample preparation
1. 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).
2. 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.
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.
1. 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 °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 8^th, 2020, the second on September 30^th, 2020, and the wastewater samples to be stored for SMF on October 14^th, 2020.
2. Stir for 30 min at 4 °C to inoculate and homogenize the Armored RNA in the samples using a magnetic stirrer.
3. Remove all particles or cells larger than 0.2 μm and perform ultrafiltration.
  1. Filter the spiked wastewater samples (120 mL from each 140 mL spiked sample) through cheesecloth and low-protein binding, 0.45 µm and 0.2 µm, 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 8^th and September 30^th were used for ultrafiltration methods with 3,000 x g and 7,500 x g, respectively.
  2. 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 µL.
  3. 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'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 30^th.
4. For SMF, perform the steps outlined below.
5. 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.
6. 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.
7. 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).
8. Stir the samples for 8 h at medium speed and allow the formed flocs to settle for another 8 h at room temperature.
9. 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 °C.
10. Carefully scrap the pellets using a sterilized spatula, and resuspend the remaining pellets in the tubes in 250 µL 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.
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 β-mercaptoethanol, according to the manufacturer's instructions, to improve the extraction efficiency. Finally, elute the RNA in 50 µL of elution buffer.
Real time-quantitative polymerase chain reaction (RT-qPCR) analysis
1. 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.
2. Design primer and probe sets using the primer design tool²⁹ and target a 95 bp region (gBlock construct) within the Armored RNA genome.
3. Obtain calibration curves for each RT-qPCR run. Include negative controls in each qPCR run. Run standards, samples, and non-template controls in triplicate.
4. Prepare each 10 µL RT-qPCR mixture in the following order: 2.5 µL of 4x master mix, 400 nM of each primer, 200 nM probe, and 2.5 µL of template, as well as ultrapure DNAse/RNAse- free distilled water.
5. Perform thermal cycling reactions at 50 °C for 5 min, followed by 45 cycles of 95 °C for 10 s and 60 °C for 30 s on a real-time PCR system. If the CT was <40, consider the sample Armored RNA positive.
6. 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.
Calculate the concentrations from the standard curve. Calculate the recovery efficiency percentage by dividing the recovered concentration by the spiked concentration.
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'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.

2. Filtration and concentration of DNA and RNA viruses for water-based epidemiology

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 °C room until processing within 24 h following sample collection.
Perform concentration of viruses from environmental samples with SMF using the steps described below.
1. Perform capsule and vacuum filtration using high-capacity groundwater sampling capsules and membrane disc filters of 0.45 µm, 0.2 µm, and 0.1 µm sizes to minimize noise during metagenomic sequencing, from larger fractions such as micro-eukaryotes and bacteria to smaller ones such as viruses.
2. 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.
Adjust the pH of the environmental surface water samples to 3.5 using 1 N HCl.
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]).
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.
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 °C.
Discard the supernatant and dissolve the resulting pellet (which theoretically contains viruses of interest) in 200 µL of 0.2 M sodium phosphate buffer.
Treat the dissolved pellet with DNase I and RNase A, following the manufacturer'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's instructions.
Extract total nucleic acids from the dissolved pellet using a DNA/RNA extraction kit, as per the manufacturer's instructions.
Quantify the total amount of extracted DNA and RNA from effluent samples using a fluorometer. Samples with concentrations >1 ng/µL are considered optimal for DNA quantification and sequencing.
Perform qPCR analysis to target enteric viruses of interest and high-throughput sequencing to identify the viral community structure.

3. Direct precipitation and concentration of microbial fractions for water-based epidemiology

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.
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.
Adjust the pH of the freshwater samples to 3.5 using 1 N HCl.
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.
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.
Discard the supernatant and resuspend the pellets with 200 µL of 0.2 M sodium phosphate buffer. Store the samples at -20 °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's instructions.
Determine the DNA nucleic acid concentration and purity with a high sensitivity fluorometer. Samples with concentrations >1 ng/µL should be optimal for DNA quantification and sequencing.

Representative Results

Evaluation of viral RNA concentration methods
All six samples processed with UF-3k x g were positive and resulted in a 13.38% ± 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% ± 2.65% recovery (Figure 1). The average recovery rates of UF-3K x g and SMF were significantly and consistently (p < 0.0001) higher than UF-7.5K x g. On the other hand, there was no significant difference (p = 0.6240) between the recovery rates of UF-3K x g and SMF. Increasing the centrifugation speed of the brand B centrifugal device to 7,500 x g from 3,000 x g resulted in lower recovery, probably due to the passage of Armored RNA through filters at an increased rate and binding of Armored RNA particles onto the filters at an increased centrifugal speed. Eliminating solid particles with ultrafiltration methods might explain the lower recovery rates of the ultrafiltration methods against SMF, considering the potential partitioning of Armored RNA to the wastewater solids³⁰. Additionally, physicochemical and biological wastewater parameters (metadata) are summarized in Table 1. Testing SMF with surface waters (step 2) resulted in a higher recovery rate, 42.7% ± 15.1%, indicating that partitioning the viruses into solid particles might have significantly affected the recovery considering low solid concentrations in surface waters.

Serial filtration and concentration of viruses
Table 3 summarizes the water quality parameters and nucleic acids extracted from environmental water samples collected within Winnipeg across three seasons. DNA was quantifiable in all sampling locations and across seasons. Supplementary Figure 1 indicates a map with the environmental surface water locations. On the other hand, The RNA values were mostly below the detection limit using a fluorometer (<0.025 ng/µL). These RNA extracts were randomly amplified to generate quantifiable cDNA for high-throughput sequencing (Table 3).

Concentration of microbial fractions from environmental water samples
Figure 2 and Figure 3 depict the workflow for the methodology and concentration of microbial fractions from environmental surface water samples. Table 4 contains the average DNA concentration, standard deviation, and the environmental conditions, such as pH, temperature, dissolved oxygen, atmospheric pressure, precipitation, and daylight times of the sampling sites of the protected (forested) and impacted (urban and agricultural) waterways analyzed in this research. Samples were collected monthly from April 2022 to October 2022 in urban and rural areas surrounding the City of Portage la Prairie along the Assiniboine river in Manitoba, Canada. The highest DNA concentration was observed in the forested site 1 and yielded 109.35-229.18 ng/µL. On the other hand, the lowest microbial DNA concentration was recovered from the agricultural site 3 and had an average concentration of 19.83-22.05 ng/µL. The average DNA concentration, standard deviation, and metadata of all the samples collected from April to October 2022 are summarized in Table 4. The DNA concentrations obtained were optimum for other downstream applications, which include PCR, qPCR, and sequencing (data not shown).

Figure 1: Percent recovery and statistical analysis for each method for wastewater samples. Abbreviations: SMF = skimmed milk flocculation; UF-3K x g = ultrafiltration at 3,000 x g; UF-7.5K x g = ultrafiltration at 7,500 x g. Means with an asterisk (*) indicate significant differences at the 0.05 level across treatments; n = 6. Please click here to view a larger version of this figure.

Figure 2: Schematic representation of the methodology used to concentrate and quantify viruses from urban waterbodies of Manitoba that receive wastewater effluents. Created with BioRender.com. Please click here to view a larger version of this figure.

Figure 3: Graphical abstract of the concentration of viruses, bacteria, and microeukaryotes for DNA quantification and sequencing. Created with BioRender.com. Please click here to view a larger version of this figure.

Supplementary Figure 1: Locations of 11 environmental water samples. Samples were collected along the Red and Assiniboine rivers in Winnipeg, Manitoba, as indicated by the red dots. Winnipeg's three major sewage treatment plants are indicated in blue as one of the sites visited, even though no sampling events occurred. The sources of the data are Google maps and Tableau software. Please click here to download this File.

Table 1: Water quality parameters in influent samples of NESTP, SESTP, and WESTP. Abbreviations: NESTP = North End Sewage Treatment Plant; SESTP = South End Sewage Treatment Plant; WESTP = West End Sewage Treatment Plant; MLD = million liters per day; TS = total solids; TSS = total suspended solids; BOD5 = 5 day biochemical oxygen demand; NH₄-N = ammonium-nitrogen; TP = total phosphorus; TOC = total organic carbon; TN = total nitrogen. Please click here to download this Table.

Table 2: Primer/probe sets of RT-qPCR assays. Please click here to download this Table.

Table 3: Red and Assiniboine rivers water quality parameters during sample collection in the Spring on May 16^th, Summer on August 27^th, and Fall on November 21^st, 2021) and fluorometer quantification of total nucleic acids (DNA and RNA). * The detection limit of the RNA kit is 0.025 ng/µL; ^ numbers represent range values. Abbreviations: DO = dissolved oxygen; BOD₅= 5 day biochemical oxygen demand; TP = total phosphorous. Please click here to download this Table.

Table 4: Assiniboine river water quality parameters during sample collection (April to October 2022). Please click here to download this Table.

Discussion

One of the critical steps in this study is the elimination of solid particles by applying a prefiltration step with 0.2 µm and 0.45 µm 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, prefiltration for SMF might be required depending on the type of downstream analysis. For targeted PCR studies, prefiltration might not be required. On the other hand, prefiltration might significantly improve viral metagenomics, since it reduces the background noise in viromes from larger fractions such as microeukaryotes and bacteria. This step is critical for sequencing so that larger genomes do not swarm the viral signal.

The sample volume that can be processed with sequential ultrafiltration is limited to 140 mL. This is an important limitation for samples with low virus counts. On the other hand, SMF can process much larger sample volumes, increasing the probability of recovering viruses.

The SMF's recovery rates are in agreement with other studies focusing on the concentration of viral particles from wastewater. Previously published papers reported similar recovery rates with a variety of test viruses. The reported mean recovery rates in these studies ranged between 3.4% and 14%¹³^,³¹^,³². The present methodology was a part of another study investigating SARS-CoV-2 in wastewater in relation to case numbers in Winnipeg³³. The SMF method described here has also been used to screen for SARS-CoV-2 RNA in oxidation lagoons from rural communities of Manitoba in our laboratory. The utility of the SMF method has also been used in combination with serial filtration (0.45 µm followed by 0.2 µm and 0.1 µm) to characterize viral communities in freshwater samples from urban settings using high-throughput sequencing in our laboratory (described in the second section of the methodology). Finally, the SMF method can also be applied to direct the precipitation of microbial communities, such as microeukaryotes, bacteria, and viruses from environmental water samples, and further characterization using deep-amplicon sequencing of the 18S rRNA and 16S rRNA genes, as well as the major capsid protein gene (g23) of viral groups such as T4-like viruses (Zambrano and Uyaguari-Díaz, unpublished data). The SMF method enables concentrating the microbial fractions present in water samples from large input volumes (i.e., 10-40 L) to relatively smaller volumes (i.e., 1-5 mL). As part of this methodology, ultrapure water is also included as a negative/background control. Once the microbial fractions are concentrated down, nucleic acid extraction can be conducted using commercial extraction kits or in-house protocols. SMF represents a versatile, relatively cheap, and easy/hands-off method to concentrate microbial fractions or viral particles, with no significant differences compared to ultrafiltration or tangential flow filtration approaches.

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

This work was supported by NSERC Alliance Covid-19 Grant (Award No. 431401363, 2020-2021, Drs. Yuan and Uyaguari-Díaz). 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 Métis Nation.

Materials

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

Riferimenti

Kumblathan, T., Liu, Y., Uppal, G. K., Hrudey, S. E., Lix, X. F. Wastewater-based epidemiology for community monitoring of SARS-CoV-2: progress and challenges. ACS Environmental Au. 1, 18-31 (2021).
Lu, D., Huang, Z., Luo, J., Zhang, X., Sha, S. Primary concentration-The critical step in implementing the wastewater based epidemiology for the COVID-19 pandemic: A mini-review. The Science of The Total Environment. 747, 141245 (2020).
Bi, Q. Insights into household transmission of SARS-CoV-2 from a population-based serological survey. Nature Communications. 12, 3643 (2021).
Day, M. Covid-19: identifying and isolating asymptomatic people helped eliminate virus in Italian village. British Medical Journal. 368, 1165 (2020).
Ing, A. J., Cocks, C., Green, J. P. COVID-19: in the footsteps of Ernest Shackleton. Thorax. 75 (8), 693-694 (2020).
Bivins, A., et al. Wastewater-based epidemiology: global collaborative to maximize contributions in the fight against COVID-19. Environmental Science & Technology. 54 (13), 7754-7757 (2020).
Medema, G., Heijnen, L., Elsinga, G., Italiaander, R., Brouwer, A. Presence of SARS-Coronavirus-2 RNA in sewage and correlation with reported COVID-19 prevalence in the early stage of the epidemic in the Netherlands. Environmental Science & Technology Letters. 7 (7), 511-516 (2020).
Thompson, J. R., et al. Making waves: Wastewater surveillance of SARS-CoV-2 for population-based health management. Water Research. 184, 116181 (2020).
Wu, F., et al. SARS-CoV-2 RNA concentrations in wastewater foreshadow dynamics and clinical presentation of new COVID-19 cases. The Science of the Total Environment. 805, 150121 (2022).
Kantor, R. S., Nelson, K. L., Greenwald, H. D., Kennedy, L. C. Challenges in measuring the recovery of SARS-CoV-2 from wastewater. Environmental Science & Technology. 55 (6), 3514-3519 (2021).
Chik, A. H. S., et al. Comparison of approaches to quantify SARS-CoV-2 in wastewater using RT-qPCR: Results and implications from a collaborative inter-laboratory study in Canada. Journal of Environmental Sciences. 107, 218-229 (2021).
Hjelmsø, M. H., et al. Evaluation of methods for the concentration and extraction of viruses from sewage in the context of metagenomic sequencing. PLoS One. 12 (1), e0170199 (2017).
Philo, S. E., et al. A comparison of SARS-CoV-2 wastewater concentration methods for environmental surveillance. The Science of the Total Environment. 760, 144215 (2021).
Ahmed, W., Harwood, V. J., Gyawali, P., Sidhu, J. P. S., Toze, S. Comparison of concentration methods for quantitative detection of sewage-associated viral markers in environmental waters. Applied and Environmental Microbiology. 81 (6), 2042-2049 (2015).
Calgua, B., et al. Detection and quantification of classic and emerging viruses by skimmed-milk flocculation and PCR in river water from two geographical areas. Water Research. 47 (8), 2797-2810 (2013).
Calgua, B., et al. Development and application of a one-step low cost procedure to concentrate viruses from seawater samples. Journal of Virological Methods. 153 (2), 79-83 (2008).
Cashdollar, J. L., Wymer, L. Methods for primary concentration of viruses from water samples: a review and meta-analysis of recent studies. Journal of Applied Microbiology. 115 (1), 1-11 (2013).
Calgua, B., et al. New methods for the concentration of viruses from urban sewage using quantitative PCR. Journal of Virological Methods. 187 (2), 215-221 (2013).
Gonzales-Gustavson, E., et al. Characterization of the efficiency and uncertainty of skimmed milk flocculation for the simultaneous concentration and quantification of water-borne viruses, bacteria and protozoa. Journal of Microbiological Methods. 134, 46-53 (2017).
Assis, A. S. F., et al. Optimization of the skimmed-milk flocculation method for recovery of adenovirus from sludge. The Science of the Total Environment. 583, 163-168 (2017).
Goncharova, E. A., et al. One-step quantitative RT-PCR assay with armored RNA controls for detection of SARS-CoV-2. Journal of Medical Virology. 93 (3), 1694-1701 (2021).
Yu, X. F., et al. Preparation of armored RNA as a control for multiplex real-time reverse transcription-PCR detection of influenza virus and severe acute respiratory syndrome coronavirus. Journal of Clinical Microbiology. 46 (3), 837-841 (2008).
Alygizakis, N., et al. Analytical methodologies for the detection of SARS-CoV-2 in wastewater: Protocols and future perspectives. Trends in Analytical Chemistry. 134, 116125 (2021).
Garcia, A., et al. Quantification of human enteric viruses as alternative indicators of fecal pollution to evaluate wastewater treatment processes. PeerJ. 10, e12957 (2022).
Gonzalez, R., et al. COVID-19 surveillance in Southeastern Virginia using wastewater-based epidemiology. Water Research. 186, 116296 (2020).
Hietala, S. K., Crossley, B. M. Armored RNA as virus surrogate in a real-time reverse transcriptase PCR assay proficiency panel. Journal of Clinical Microbiology. 44 (1), 67-70 (2006).
Uyaguari-Diaz, M. I., et al. A comprehensive method for amplicon-based and metagenomic characterization of viruses, bacteria, and eukaryotes in freshwater samples. Microbiome. 4 (1), 20 (2016).
Meena, G. S., Singh, A. K., Gupta, V. K., Borad, S., Parmar, P. T. Effect of change in pH of skim milk and ultrafiltered/diafiltered retentates on milk protein concentrate (MPC70) powder properties. Journal of Food Science and Technology. 55 (9), 3526-3537 (2018).
. Geneious Available from: https://www.geneious.com (2021)
Ye, Y., Ellenberg, R. M., Graham, K. E., Wigginton, K. R. Survivability, partitioning, and recovery of enveloped viruses in untreated municipal wastewater. Environmental Science & Technology. 50 (10), 5077-5085 (2016).
Philo, S. E., et al. Development and validation of the skimmed milk pellet extraction protocol for SARS-CoV-2 wastewater surveillance. Food and Environmental Virology. 14 (4), 355-363 (2022).
Monteiro, S., et al. Recovery of SARS-CoV-2 from large volumes of raw wastewater is enhanced with the inuvai R180 system. Journalof Environmental Management. 304, 114296 (2022).
Yanaç, K., Adegoke, A., Wang, L., Uyaguari, M., Yuan, Q. Detection of SARS-CoV-2 RNA throughout wastewater treatment plants and a modeling approach to understand COVID-19 infection dynamics in Winnipeg, Canada. The Science of The Total Environment. 825, 153906 (2022).

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Yanaç, K., Francis, J., Zambrano-Alvarado, J., Yuan, Q., Uyaguari-Díaz, M. Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration. J. Vis. Exp. (193), e65058, doi:10.3791/65058 (2023).