key: cord-0824769-lgllny3m
authors: Rondeau, Nicole C.; Rose, Oliver J.; Ariyan, Lina A.; Mailloux, Brian J.; Miranda, JJ L.
title: Accessible and Validated Processing of SARS-CoV-2 from Wastewater
date: 2021-05-27
journal: Microbiology resource announcements
DOI: 10.1128/mra.00174-21
sha: 6a9277d311595367532b7832c867e17a6768ec56
doc_id: 824769
cord_uid: lgllny3m

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), is shed into wastewater. Accessible methods are necessary for processing samples in a myriad of contexts. We optimized a protocol for extracting viral RNA for downstream experiments. Our pipeline was validated with SARS-CoV-2 itself as a matrix recovery and quantitative measurement control.

Basel, Switzerland) performed the CDC-recommended amplification (7) , as follows: 45°C for 2 min, 95°C for 2 min, 45 cycles of 95°C for 3 s with 55°C for 30 s, and 40°C for 30 s. Cycle threshold (C T ) values were calculated by LightCycler 480 software (Roche). We determined a standard curve using quantitative PCR (qPCR) control RNA from heat-inactivated SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources). Spike-in experiments were conducted only with samples that tested negative.

Our protocol was validated using SARS-CoV-2 instead of a substitute. This was not possible with methods (2) used early in the pandemic, prior to the availability of SARS-CoV-2 controls. The limit of detection was determined by spike-in experiments with 100 genome equivalents/ml, which consistently yielded positive-result C T values of ,40 ( Table 1) . Observation of less than the expected ;3.32-C T value increase in many FIG 1 Accessible preparation of SARS-CoV-2 from wastewater for RNA analysis. Raw wastewater is processed with a 20-mm filter to remove large debris, slow centrifugation at 2,600 Â g to remove small debris and bacteria, a 0.22-mm filter to remove bacteria, a 100-kDa centrifugal filter to remove small molecules, and spin column RNA purification to remove DNA. The resulting viral RNA fraction is suitable for subsequent PCR testing. Percentages on the left indicate the recovery of 10,000 SARS-CoV-2 genome equivalents/ml initial volume spiked in before the indicated processing steps. Numbers represent the mean and standard deviation of four independent biological replicates. RNA samples diluted 1:10 for RT-qPCR suggests the lingering presence of PCR inhibitors commonly found in wastewater. For more detailed evaluation of protocol efficiency, we spiked in 10,000 genome equivalents/ml initial volume at different steps. We used C T values from RNA samples diluted 1:10 and measured recovery of ;1% of SARS-CoV-2 processed through the entire protocol. Parallel spike-in experiments at different steps identified concentration by centrifugal ultrafiltration as a critical procedure during which virus yield is reduced ;10-fold (Fig. 1) . We noted significant variability between samples, likely due to varied wastewater composition among sites and days. Overall, the protocol demonstrates sensitivity and efficiency suitable for accessible wastewater-based epidemiology. We hope that this simplified procedure provides a template for making experiments with or monitoring of SARS-CoV-2 in wastewater accessible to a broad audience. The protocol may also potentially be used to study other viruses or metagenomes.

SARS-CoV-2 in wastewater: state of the knowledge and research needs

Sewage analysis as a tool for the COVID-19 pandemic response and management: the urgent need for optimised protocols for SARS-CoV-2 detection and quantification

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

Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater

First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: a proof of concept for the wastewater surveillance of COVID-19 in the community

Heat inactivation of different types of SARS-CoV-2 samples: what protocols for biosafety, molecular detection and serological diagnostics?

Centers for Disease Control and Prevention. 2020. CDC 2019-novel coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel: instructions for use

We thank the President's Office at Barnard College for supporting this project. We are grateful to Susan K. De Long (Colorado State University) and Duane Graves (Geosyntec Consultants) for helpful discussions.The following reagents were deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-WA1/2020, Heat Inactivated, NR-52286 and Quantitative PCR (qPCR) Control RNA from Heat-Inactivated SARS-Related Coronavirus 2, Isolate USA-WA1/2020, NR-52347.