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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 L mL-1 sample, and an elution temperature of 60 {degrees}C. The performance of this optimized method was further assessed in recovery experiments using wastewater samples spiked at 1.8x106 and 1.8x104 gene copies L-1 (GU L-1) with Gamma Inactivated SARS-COV-2 (GI-SCV-2) and 1.0x106 and 1.0x104 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 4.6x104 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.