Development and optimization of a new method for direct extraction of SARS-CoV-2 RNA from municipal wastewater using magnetic beads

The use of magnetic beads in the extraction of nucleic acids from wastewater is presented as an approach to simplify extraction techniques for the detection of SARS-CoV-2 viral fragments in wastewater. In particular, this paper describes the development and optimization of a direct method for extracting SARS-CoV-2 RNA from municipal wastewater using magnetic beads. The recovery efficiency of the method using Accuplex SARS-CoV-2 Positive Reference Material (ASCV-2) was examined. Method factors assessed were sample volume, concentration of magnetic bead mix, elution temperature, and water matrix (deionized (DI) water and wastewater). The combination of optimized method parameters that resulted in the highest RNA recovery in both DI water (26.0 ± 0.8%) and wastewater (11.8 ± 1.4%) was a sample volume of 1.0 mL, a magnetic beads concentration of 100 µL mL-1 sample, and an elution temperature of 60 °C. The performance of this optimized method was further assessed in recovery experiments using wastewater samples spiked at 1.8×106 and 1.8×104 gene copies L-1 (GU L-1) with Gamma Inactivated SARS-COV-2 (GI-SCV-2) and 1.0×106 and 1.0×104 infectious units L-1 of Human Coronavirus 229E (HCV 229E) as viral surrogates. Recoveries of 86.1 and 4.6% were achieved for wastewater samples spiked with GI-SCV-2 at low and high concentrations, respectively. In assessing the effects of wastewater pre-filtration and addition of DL-Dithiothreitol (DTT, used to inactivate RNases that may degrade RNA) on recovery efficiency of ASCV-2, the magnetic bead-based extraction protocol performed optimally with unfiltered wastewater without DTT (recovery = 17.4 ± 0.4%). The method limit of detection (MLOD) for ASCV-2 recovered from pre-filtered wastewater was determined to be 4.6×104 GU L-1 (95% degree of confidence). Using this optimized magnetic bead-based extraction protocol, the presence of SARS-CoV-2 RNA was verified in wastewater collected from sewershed locations in Atlantic Canada. This emerging RNA extraction method is direct, rapid, and does not require the use of specialized equipment, thus offering advantageous application for laboratories with limited resources. As such, this method is an indispensable tool in the monitoring of wastewater for SARS-CoV-2 to potentially understand COVID-19 infection occurrence within communities and inform public health leaders. View this table: ### Competing Interest Statement The authors declare that LuminUltra Technologies Ltd. provided equipment, some reagents and technical support to assist in this work. The authors declare that they do not have any financial interest in LuminUltra Technologies Ltd. This study was funded through support from an NSERC COVID-19 Alliance Grant [grant number ALLRP 554503-20], an NSERC Collaborative Research and Development Grant in partnership with Halifax Water [grant number CRDPJ 539387-19] and the NSERC/Halifax Water Industrial Research Chair program [grant number IRCPJ: 349838-16]. ### Funding Statement This study was funded through support from an NSERC COVID-19 Alliance Grant [grant number ALLRP 554503-20], an NSERC Collaborative Research and Development Grant in partnership with Halifax Water [grant number CRDPJ 539387-19] and the NSERC/Halifax Water Industrial Research Chair program [grant number IRCPJ: 349838-16]. Wastewater was spiked at the National Microbiology Laboratory in Winnipeg with GI-SCV-2 and HCV 229E acquired from the University of Alberta through the Canadian Water Network COVID-19 Coalition. The authors thank LuminUltra Technologies for providing qPCR technology and for technical support that was led through Dr. Jordan Schmidt throughout the project. The authors would like to extend thanks to municipal staff in communities A, B, and C for sample collection. As well, the authors would like to extend thanks to researchers Paul Bjorndahl and Sebastian Munoz from the Centre for Water Resources Studies at Dalhousie University for providing technical reviews, data analysis, and data visualization during the study. Graphical abstract was adapted from Quantifying SARS-CoV-2 Virions in City Wastewater, by BioRender.com (2020); retrieved from . ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Not applicable All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All applicable data available in manuscript text