TY - JOUR

T1 - Concentration and quantification of SARS-CoV-2 RNA in wastewater using polyethylene glycol-based concentration and qRT-PCR

AU - Farkas, Kata

AU - Hillary, Luke

AU - Thorpe, Jamie

AU - Walker, David

AU - Lowther, James A.

AU - McDonald, James

AU - Malham, Shelagh

AU - Jones, Davey L.

PY - 2021/2/23

Y1 - 2021/2/23

N2 - 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.

AB - 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.

KW - COVID-19

KW - public health

KW - wastewater virology

KW - Sewage

KW - environmental sample

U2 - 10.3390/mps4010017

DO - 10.3390/mps4010017

M3 - Article

C2 - 33672247

VL - 4

JO - Methods and Protocols

JF - Methods and Protocols

SN - 2409-9279

IS - 1

M1 - 17

ER -