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

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...

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Autores principales: Farkas, Kata, Hillary, Luke S., Thorpe, Jamie, Walker, David I., Lowther, James A., McDonald, James E., Malham, Shelagh K., Jones, Davey L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005995/
https://www.ncbi.nlm.nih.gov/pubmed/33672247
http://dx.doi.org/10.3390/mps4010017
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author Farkas, Kata
Hillary, Luke S.
Thorpe, Jamie
Walker, David I.
Lowther, James A.
McDonald, James E.
Malham, Shelagh K.
Jones, Davey L.
author_facet Farkas, Kata
Hillary, Luke S.
Thorpe, Jamie
Walker, David I.
Lowther, James A.
McDonald, James E.
Malham, Shelagh K.
Jones, Davey L.
author_sort Farkas, Kata
collection PubMed
description 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.
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spelling pubmed-80059952021-03-30 Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR Farkas, Kata Hillary, Luke S. Thorpe, Jamie Walker, David I. Lowther, James A. McDonald, James E. Malham, Shelagh K. Jones, Davey L. Methods Protoc Protocol 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. MDPI 2021-02-23 /pmc/articles/PMC8005995/ /pubmed/33672247 http://dx.doi.org/10.3390/mps4010017 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ).
spellingShingle Protocol
Farkas, Kata
Hillary, Luke S.
Thorpe, Jamie
Walker, David I.
Lowther, James A.
McDonald, James E.
Malham, Shelagh K.
Jones, Davey L.
Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR
title Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR
title_full Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR
title_fullStr Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR
title_full_unstemmed Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR
title_short Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR
title_sort concentration and quantification of sars-cov-2 rna in wastewater using polyethylene glycol-based concentration and qrt-pcr
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005995/
https://www.ncbi.nlm.nih.gov/pubmed/33672247
http://dx.doi.org/10.3390/mps4010017
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