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Protocol to longitudinally quantify SARS-CoV-2 RNA in wastewater using RT-qPCR and pepper mild mottle virus
Bryan Sanchez Jimenez1, Trinity Sterling2, Austin Brown2
1Department of Health Sciences, Stetson University, 421 N Woodland Boulevard, DeLand, FL 32723, USA; Department of Microbiology and Immunology, New York Medical College, 40 Sunshine Cottage Road, Valhalla, NY 10595, USA.
None:
Wastewater surveillance allows severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection levels to be tracked in a community. Here, we present a protocol to longitudinally quantify SARS-CoV-2 RNA in wastewater using quantitative reverse-transcription PCR (RT-qPCR) and pepper mild mottle virus (PMMoV) normalization. We describe steps for the pasteurization of wastewater samples, solids separation, supernatant filtration, viral precipitation and concentration, and RNA extraction. We then detail procedures for RT-qPCR, viral concentration extrapolation, PMMoV normalization, and longitudinal analysis. This protocol has the potential to be used for surveillance of other microorganisms. For complete details on the use and execution of this protocol, please refer to Sanchez Jimenez et al.1.
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