Protocol to longitudinally quantify SARS-CoV-2 RNA in wastewater using RT-qPCR and pepper mild mottle virus normalization

Bryan Sanchez Jimenez; Trinity Sterling; Austin Brown; Brian Modica; Kaylee Gibson; Hannah Collins; Carolyn Koch; Tyler Schwarz; Kristine N Dye

doi:10.1016/j.xpro.2024.103001

Protocol to longitudinally quantify SARS-CoV-2 RNA in wastewater using RT-qPCR and pepper mild mottle virus normalization

STAR Protoc. 2024 Apr 9;5(2):103001. doi: 10.1016/j.xpro.2024.103001. Online ahead of print.

Authors

Bryan Sanchez Jimenez¹, Trinity Sterling², Austin Brown², Brian Modica², Kaylee Gibson², Hannah Collins², Carolyn Koch², Tyler Schwarz², Kristine N Dye³

Affiliations

¹ Department 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. Electronic address: bjimenez4@student.nymc.edu.
² Department of Health Sciences, Stetson University, 421 N Woodland Boulevard, DeLand, FL 32723, USA.
³ Department of Health Sciences, Stetson University, 421 N Woodland Boulevard, DeLand, FL 32723, USA; Department of Biology, Stetson University, 421 N Woodland Boulevard, DeLand, FL 32723, USA. Electronic address: kdye1@stetson.edu.

Abstract

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.

Keywords: Environmental sciences; Microbiology; Molecular Biology.