Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR

• Published in: Methods and protocols Vol. 4; no. 1; p. 17
• Main Authors: Farkas, Kata, Hillary, Luke S., Thorpe, Jamie, Walker, David I., Lowther, James A., McDonald, James E., Malham, Shelagh K., Jones, Davey L.
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 23.02.2021; MDPI
• Subjects: Chemical properties; Coronaviruses; COVID-19; DNA viruses; environmental samples; Epidemiology; Genetic transcription; Health aspects; Organic matter; Physicochemical properties; Polyethylene glycol; Polymerase chain reaction; Protocol; public health; Reverse transcription; Ribonucleic acid; RNA; RNA viruses; Severe acute respiratory syndrome coronavirus 2; Sewage; Viral diseases; Virus diseases; Viruses; Wastewater; wastewater virology; United Kingdom; St Louis Missouri; France; United States > US; Germany; COVID-19; environmental samples; wastewater virology; public health; sewage
• ISSN: 2409-9279; 2409-9279
• DOI: 10.3390/mps4010017

Wastewater-based epidemiology has become an important tool for the surveillance of SARS-CoV-2 outbreaks. However, the detection of viruses in sewage is challenging and to date there is no standard method available which has been validated for the sensitive detection of SARS-CoV-2. In this paper, we describe a simple concentration method based on polyethylene glycol (PEG) precipitation, followed by RNA extraction and a one-step quantitative reverse transcription PCR (qRT-PCR) for viral detection in wastewater. PEG-based concentration of viruses is a simple procedure which is not limited by the availability of expensive equipment and has reduced risk of disruption to consumable supply chains. The concentration and RNA extraction steps enable 900–1500× concentration of wastewater samples and sufficiently eliminates the majority of organic matter, which could inhibit the subsequent qRT-PCR assay. Due to the high variation in the physico-chemical properties of wastewater samples, we recommend the use of process control viruses to determine the efficiency of each step. This procedure enables the concentration and the extraction the DNA/RNA of different viruses and hence can be used for the surveillance of different viral targets for the comprehensive assessment of viral diseases in a community.