Development of a rapid pre-concentration protocol and a magnetic beads-based RNA extraction method for SARS-CoV-2 detection in raw municipal wastewater

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.