Sewage, Salt, Silica, and SARS-CoV-2 (4S): An Economical Kit-Free Method for Direct Capture of SARS-CoV-2 RNA from Wastewater

Abstract

Wastewater-based epidemiology is an emerging tool to monitor COVID-19 infection levels by measuring the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. There remains a need to improve wastewater RNA extraction methods’ sensitivity, speed, and reduce reliance on often expensive commercial reagents to make wastewater-based epidemiology more accessible. We present a kit-free wastewater RNA extraction method, titled “Sewage, Salt, Silica and SARS-CoV-2” (4S), that employs the abundant and affordable reagents sodium chloride (NaCl), ethanol, and silica RNA capture matrices to recover sixfold more SARS-CoV-2 RNA from wastewater than an existing ultrafiltration-based method. The 4S method concurrently recovered pepper mild mottle virus (PMMoV) and human 18S ribosomal subunit rRNA, which have been proposed as fecal concentration controls. The SARS-CoV-2 RNA concentrations measured in three sewersheds corresponded to the relative prevalence of COVID-19 infection determined via clinical testing. Lastly, controlled experiments indicate that the 4S method prevented RNA degradation during storage of wastewater samples, was compatible with heat pasteurization, and in our experience, 20 samples can be processed by one lab technician in approximately 2 h. Overall, the 4S method is promising for effective, economical, and accessible wastewater-based epidemiology for SARS-CoV-2, providing another tool to fight the global pandemic.

Tools for interpretation of wastewater SARS-CoV-2 temporal and spatial trends demonstrated with data collected in the San Francisco Bay Area

“…More recently, four biological markers have emerged as promising candidates to normalize SARS-CoV-2 RNA signal for fecal content. Pepper mild mottle virus (PMMoV), a nonenveloped RNA plant virus, is commonly used for COVID-19 WBE ( D'Aoust et al, 2021b ; Feng et al, 2021 ; Whitney et al, 2021 ; Wu et al, 2020 ) but concentrations in sewage vary with season and local diet ( Symonds et al, 2019 ). Another normalization biomarker is the cross-assembly phage (crAssphage), a non-enveloped, DNA virus that ubiquitously infects the human gut commensal bacteria Bacteroides ( Edwards et al, 2019 ; Green et al, 2020 ; Stachler et al, 2017 ; Wilder et al, 2021 ).…”

“…In addition, Bacteroides HF183 16S rRNA gene is widely used for detecting fecal contamination in environmental waters ( Green et al, 2020 ; Shanks et al, 2008 ), and recent studies ( D'Aoust et al, 2021b ; Kapoor et al, 2015 ; Pitkänen et al, 2013 ) have targeted HF183 rRNA (versus the rRNA gene) to increase the sensitivity of the assay ( D'Aoust et al, 2021b ; Feng et al, 2021 ). Lastly, the human 18S ribosomal subunit RNA (18S rRNA) assay has been proposed as a normalization biomarker because it targets human cells that are shed in feces ( D'Aoust et al, 2021b ; Whitney et al, 2021 ). While each of these normalization biomarkers has been assessed independently, they have not all been compared within the same study.…”

First detection of SARS-CoV-2 proteins in wastewater samples by mass spectrometry

“…In contrast, after LC-MS/MS analyses of nasal swabs and gargle samples collected from positive COVID-19 patients, the most abundant peptides were from structurally abundant proteins (N most often, then the E, M, and S proteins) [25][26][27]. Those structural proteins are all part of the viral envelope, which makes them the easiest to detect in a freshly collected clinical sample, but they are also the proteins that are most-exposed to environmental factors, and will likely degrade first in wastewaters known to contain many chemicals with the potential to degrade SARS-CoV-2 [28].…”

Interpretation of temporal and spatial trends of SARS-CoV-2 RNA in San Francisco Bay Area wastewater

“…More recently, four biological markers have emerged as promising candidates to normalize SARS-CoV-2 RNA signal for fecal content. Pepper mild mottle virus (PMMoV), a nonenveloped RNA plant virus, is commonly used for COVID-19 WBE (D'Aoust et al, 2021b;Feng et al, 2021;Whitney et al, 2021;Wu et al, 2020) but concentrations in sewage vary with season and local diet (Symonds et al, 2019). Another normalization biomarker is the cross-assembly phage (crAssphage), a non-enveloped, DNA virus that ubiquitously infects the human gut commensal bacteria Bacteroides (Edwards et al, 2019;Green et al, 2020;Stachler et al, 2017;Wilder et al, 2021).…”