ABSTRACT
In developing an effective monitoring program for the wastewater surveillance of SARS-CoV-2 ribonucleic acid (RNA), the importance of sampling methodology is paramount. Passive sampling has been shown to be an effective tool to detect SARS-CoV-2 RNA in wastewater. However, the adsorption characteristics of SARS-CoV-2 RNA on passive sampling material are not well-understood, which further obscures the relationship between wastewater surveillance and community infection. In this work, adsorption kinetics and equilibrium characteristics were evaluated using batch-adsorption experiments for heat-inactivated SARS-CoV-2 (HI-SCV-2) adsorption to electronegative filters. Equilibrium isotherms were assessed or a range of total suspended solids (TSS) concentrations (118, 265, and 497 mg L-1) in wastewater, and a modeled q(max) of 7 X 10(3) GU cm(-2) was found. Surrogate adsorption kinetics followed a pseudo-first-order model in wastewater with maximum concentrations achieved within 24 h. In both field and isotherm experiments, equilibrium behavior and viral recovery were found to be associated with wastewater and eluate TSS. On the basis of the results of this study, we recommend a standard deployment duration of 24-48 h and the inclusion of eluate TSS measurement to assess the likelihood of solids inhibition during analysis.
ABSTRACT
In this work, a rapid and simplified method for extracting SARS-CoV-2 RNA from whole wastewater using a magnetic beads-based protocol is presented. The described method involves the centrifugation of a 50-mL aliquot of raw wastewater influent for 5 min to obtain a 500-μL pellet, which is eluted with 2 mL of a Tween®20-based elution buffer;1 mL of the elute is extracted for RNA using a direct magnetic bead-based extraction method. RNA recovery was examined in several bench-scale experiments using heat-inactivated SARS-CoV-2 (HI-SCV-2) spiked into raw wastewater to assess the effects of different solids pellet : buffer ratios, inhibition mitigation strategies, and varying levels of total suspended solids. When the method was assessed using an influent wastewater sample known to contain SARS-CoV-2, the viral signal was detected in all five biological replicates, whereas direct extraction of 1-mL aliquots of the raw wastewater resulted in a 40% viral detection rate. The experimental method limit of detection (MLOD) using HI-SCV-2 spiked into raw wastewater was 50 GU mL−1 with a 95% limit of detection. Using the described protocol, the presence of SARS-CoV-2 RNA was verified in wastewater collected from wastewater treatment facilities (WWTFs) in Atlantic Canada over a period of 15 weeks during the rise and fall of a COVID-19 outbreak. This method is effective and rapid and could provide potential application for laboratories with limited resources. Of approximately 50 methods that have been developed for measuring SARS-CoV-2 in wastewater referenced in the literature, this is the first to advance a robust magnetic beads-based RNA extraction technique from whole wastewater without extensive sample pre-treatment. The novel application of this method in the rapid extraction of SARS-CoV-2 RNA from municipal wastewater is an indispensable tool to potentially understand COVID-19 infection occurrence within communities.
ABSTRACT
The overall objective of this work was to develop a simple and effective passive sampling protocol for the detection of SARS-CoV-2 in sewer catchments at targeted institutional-level sampling sites in a region of low COVID-19 prevalence. We developed a new 3D-printed sampling cage and assessed four commercially-available materials (cotton gauze, cotton cheesecloth, cellulose sponges, and electronegative filters) for RNA adsorption in the cage. We determined that cheesecloth and electronegative filters provided an effective approach for collecting and measuring SARS-CoV-2 in wastewater. We also compared the performance of three elution mixtures (a commercially-available lysis buffer, a Tween (R) 20-based buffer, and a 1:1 acetonitrile:water mixture) for detection of heat-inactivated SARS-CoV-2 reference material (HI-SCV-2) spiked into municipal wastewater at 1.0 x 10(3) genomic units per millilitre (GU mL(-1)). The highest mean RNA concentrations were achieved using the cheesecloth (7.0 x 10(4) +/- 3.7 x 10(4) GU per eluate) and electronegative filters (2.3 x 10(4) +/- 2.5 x 10(4) GU per eluate) in combination with the Tween (R) 20-based buffer with positive detections in all three biological replicates for both material types. We deployed passive samplers at two sewer catchments (locations A and B) to compare the performance of each passive sampler material type in the field. Over 15 sampling events at each site, we demonstrated that both cheesecloth (location A) and electronegative filters (location B) coupled with a Tween (R) 20-based elution technique could be utilized for the reliable detection of SARS-CoV-2. These results have demonstrated a quick and effective passive sampling approach for SARS-CoV-2 detection in targeted locations in wastewater collection systems, which may have long-term applicability as global vaccination programs evolve.