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. 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.
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.
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…
The authors have nothing to disclose.
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.
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 |