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Method Article

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

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DOI:

10.3791/65058

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March 17th, 2023

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

In This Article

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.

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Protocol

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

  1. 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.
  2. 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 8th, 2020, the second on September 30th, 2020, and the wastewater samples to be stored for SMF on October 14th, 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 8th and September 30th 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 30th.
    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.
  3. 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.
  4. 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.
  5. Calculate the concentrations from the standard curve. Calculate the recovery efficiency percentage by dividing the recovered concentration by the spiked concentration.
  6. 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

  1. 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.
  2. 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.
  3. Adjust the pH of the environmental surface water samples to 3.5 using 1 N HCl.
  4. 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]).
  5. 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.
  6. 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.
  7. Discard the supernatant and dissolve the resulting pellet (which theoretically contains viruses of interest) in 200 µL of 0.2 M sodium phosphate buffer.
  8. 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.
  9. Extract total nucleic acids from the dissolved pellet using a DNA/RNA extraction kit, as per the manufacturer's instructions.
  10. 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.
  11. 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

  1. 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.
  2. 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.
  3. Adjust the pH of the freshwater samples to 3.5 using 1 N HCl.
  4. 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.
  5. 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.
  6. 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.
  7. 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.

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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 (...

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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, prefiltrat...

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Disclosures

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.

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.2 M sodium phosphate buffer with a pH 7.5Alfa AesarJ62041APFisher Scientific, Fair Lawn, NJ, USA
0.2 μm 47-mm Supor-200 membrane disc filtersVWR66234Pall Corporation, Ann Arbor, MI
0.45 μm 47-mm Supor-200 membrane disc filtersVWR60043Pall Corporation, Ann Arbor, MI
4X TaqMan Fast Virus 1-Step Master MixThermo Fisher Scientific4444432Life Technologies, Carlsbad, CA, USA
Armored RNA Quant IPC-1 Processing ControlAsuragen49650Asuragen, Austin, TX, USA
Brand A, Jumbosep Centrifugal Device, 30-kDaPall OD030C65Pall Corporation, Ann Arbor, MI
Brand B, Microsep Advance Centrifugal Device, 30-kDaPallMCP010C46Pall Corporation, Ann Arbor, MI
Centrifuge tubes (50 ml) Nalgene3119-0050PKThermo Fisher Scientific
DNAse IInvitrogen18047019Thermo Fisher Scientific
Dyna Mag-2Invitrogen12027Thermo Fisher Scientific
GWV High Capacity Groundwater Sampling Capsules - 0.45 µmPall12179Pall Corporation, Ann Arbor, MI
Hydrochloric acid, 1N standard solutionThermo Fisher ScientificAC124210025Fisher Scientific, Fair Lawn, NJ, USA
MagMAX Microbiome Ultra Nucleic Acid Isolation KitApplied biosystemsA42358Thermo Fisher Scientific
Nuclease free waterPromegaP1197Promega Corporation, Fitchburg, WI, USA
Peristaltic pumpMasterflex, Cole-Parmer instrument7553-20Thermo Fisher Scientific
pH meter Denver instrumentRK-59503-25Cole-Parmer. This product has been discontinued
Phenol:chloroform:isoamyl alcohol 25:24:1Invitrogen15593031Fisher Scientific, Fair Lawn, NJ, USA
Primers and probe setsIDTIntegrated DNA Technologies, Inc., Coralville, IA, USA
Qiagen All-prep DNA/RNA power microbiome kitQiagenQiagen Sciences, Inc., Germantown, MD, USA
QuantStudio 5 Real-Time PCR SystemThermo Fisher ScientificA34322Life Technologies, Carlsbad, CA, USA
Qubit 1X dsDNA High Sensitivity (HS) assay kitInvitrogenQ33231Thermo Fisher Scientific
Qubit 4 Fluorometer, with WiFiInvitrogenQ33238Thermo Fisher Scientific
Qubit RNA High Sensitivity (HS) assay kitInvitrogenQ32855Thermo Fisher Scientific
RNAse AInvitrogenEN0531Thermo Fisher Scientific
RNeasy PowerMicrobiome KitQiagen26000-50Qiagen Sciences, Inc., Germantown, MD, USA
Skim milk powderDifco (BD Life Sciences)DF0032173Fisher Scientific, Fair Lawn, NJ, USA
Sodium phosphate bufferAlfa AesarAlfa Aesar, Ottawa, ON, Canada
Synthetic seawaterVWR RC8363-1RICCA chemical company
Synthetic single-stranded DNA gBlockIDTIntegrated DNA Technologies, Inc., Coralville, IA, USA
VacuCap 90 Vacuum Filtration Devices - 0.1 µm, 90 mm, gamma-irradiatedPall4621Pall Corporation, Ann Arbor, MI
VacuCap 90 Vacuum Filtration Devices - 0.2 µm, 90 mm, gamma-irradiatedPall4622Pall Corporation, Ann Arbor, MI
β-mercaptoethanolGibco21985023Fisher Scientific, Fair Lawn, NJ, USA

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Virus ConcentrationUltrafiltration MethodEnvironmental Water SamplesWastewater EpidemiologyArmored RNAPrefiltration TechniqueViral RNA ExtractionRT qPCR AnalysisMicrobial Recovery
Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration
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