Correction: Farkas et al. Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR. Methods Protoc. 2021, 4, 17 (original) (raw)
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Methods and Protocols, 2021
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 reco...
protocols.io, 2021
The following protocol describes our workflow for processing wastewater with the goal of detecting the genetic signal of SARS-CoV-2. The steps include pasteurization, virus concentration, RNA extraction, and quantification by RT-qPCR. We include auxiliary steps that provide new users with tools and strategies that will help troubleshoot key steps in the process. This protocol is one of the safest, cheapest, and most reproducible approaches for the detection of SARS-CoV-2 RNA in wastewater. Furthermore, the RNA obtained using this protocol, minus the pasteurization step, can be sequenced both using a targeted approach sequencing specific regions or the whole genome. The protocol was adopted by the New York City Department of Environmental Protection in August 2020 to support their efforts in monitoring SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. Owing to a pasteurization step, it is safe for use in a BSL1+ facility. This step also increases the genetic signal of the virus while making the protocol safe for the personnel involved. This protocol could be used to isolate a variety of other clinically relevant viruses from wastewater and serve as a foundation of a wastewater surveillance strategy for monitoring community spread of known and emerging viral pathogens. .
Science of The Total Environment
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Journal of Biomolecular Techniques : JBT, 2021
Wastewater surveillance for monitoring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important epidemiologic tool for the assessment of population-wide coronavirus disease 2019 (COVID-19). This tool can be successfully implemented only if SARS-CoV-2 RNA in wastewater can be accurately recovered and quantified. The lack of standardized procedure for wastewater virus analysis has resulted in varying SARS-CoV-2 concentrations for the same sample. This study reports the effect of 4 key factors-sample volume, percentage polyethylene glycol (PEG)-NaCl, incubation period, and storage duration at 4 Con the recovery of spiked noninfectious SARS-CoV-2 RNA in raw sewage and sludge samples. N1 and N2 genes of SARS-CoV-2 were quantified using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and digital droplet PCR (RT-ddPCR) techniques. Results indicate that 1) for raw sewage, 50-ml sample volume, 30% PEG-NaCl addition, 6-h incubation, and sample analysis within 24 h of collection can result in much better RNA recovery (RT-qPCR: 72% for N1 and 82% for N2; RT-ddPCR: 55% for N1 and 85% for N2) when compared with commonly used PEGbased method; 2) for sludge, the sample analysis using raw sewage protocol and all other variations of each factor mostly resulted in false negatives for both N1 and N2. The absence of N1 and N2 suggests that sludge samples probably need a pretreatment step that releases RNA entrapped in sludge solids back into bulk solution. In conclusion, our modified PEG-based concentration method can cut down the analysis time at least by half, which in turn helps to implement early detection system for SARS-CoV-2 in wastewater.
Efficient detection of SARS-CoV-2 RNA in the solid fraction of wastewater
Science of The Total Environment, 2021
SARS-CoV-2 RNAs in wastewater from a manhole and two WWTPs in Japan were assessed. • Four virus recovery methods were compared for enveloped SARS-CoV-2 detection. • SARS-CoV-2 RNAs were efficiently detected in the solid fraction of the wastewater. • Detection sensitivity was higher when the duplex RT-qPCR assay was used. • Viral RNA concentrations showed correlation with number of cases in high prevalence areas.
medRxiv (Cold Spring Harbor Laboratory), 2021
Wastewater-based epidemiology has gained attention throughout the world for detection of SARS-CoV-2 RNA in wastewater to supplement clinical testing. Methods have been developed using both the liquid and the solid fraction of wastewater, with some studies reporting higher concentrations in solids. To investigate this relationship further, we collaborated with six other laboratories to conduct a study across five publicly owned treatment works (POTWs) where both primary solids and raw wastewater influent samples were collected and quantified for SARS-CoV-2 RNA. Solids and influent samples were processed by participating laboratories using their respective methods and retrospectively paired based on date of collection. SARS-CoV-2 RNA concentrations by mass (gene copies per gram) were higher in solids than in influent by approximately three orders of magnitude. Concentrations in matched solids and influent were positively and significantly correlated at all five POTWs. RNA concentrations in both solids and influent were correlated to COVID-19 incidence rates in the sewershed and thus representative of disease burden; the solids methods appeared to produce a comparable relationship between SARS-CoV-2 RNA concentration measurements and incidence rates across all POTWs. Solids and influent methods showed comparable sensitivity, N gene detection frequency, and calculated empirical incidence rate lower limits. Analysis of solids has the advantage of using less sample volume to achieve similar sensitivity to influent methods. .
Tracking SARS-CoV-2 RNA through the Wastewater Treatment Process
ACS ES&T Water
Municipal sewage carries degraded and intact viral particles and RNA (ribonucleic acid) of SARS-CoV-2 (severe acute respiratory coronavirus 2), shed by COVID-19 (coronavirus disease 2019) patients, to sewage and eventually to wastewater treatment plants. Proper wastewater treatment can prevent uncontrolled discharges of the virus into the environment. However, the role of different wastewater treatment stages in reducing viral RNA concentrations is, thus far, unknown. Here, we quantified SARS-CoV-2 RNA in raw sewage and during the main stages of the activated sludge process from two wastewater treatment plants in Israel, on three different days during the 2020 COVID-19 outbreak. To reduce the detection limit, samples were concentrated prior to quantification by real-time polymerase chain reaction by a factor of 2−43 using ultrafiltration. On average, ∼1 log RNA removal was attained by each of the primary and secondary treatment steps; however, >100 copies of SARS-CoV-2 RNA/ mL remained in the secondary effluents. Following chlorination, SARS-CoV-2 RNA was detected only once, likely due to an insufficient chlorine dose. Our results emphasize the capabilities and limitations of the conventional wastewater treatment process in reducing the SARS-CoV-2 RNA concentration and present preliminary evidence for the importance of tertiary treatment and chlorination in reducing dissemination of the virus to the environment.
Flora the Journal of Infectious Diseases and Clinical Microbiology, 2021
SARS-CoV-2 RNA'nın Basit bir Konsantrasyon Yöntemiyle Tespit Edilmesi: Çorum'da Pilot Çalışma Sabiha AYDOĞDU 1 (İD), Djursun Karasartova 2 (İD), Ünsal savCI 2 (İD), Ayşe Semra GÜRESER 2 (İD), Gönül ARSlAn AkvERAn 3 (İD), Merve aKtI 2 (İD), Büşra GÜREl 2 (İD), Çağdaş KoCaman 2 (İD), aytaç aCar 2 (İD), nezahat kOŞAR 2 (İD), Lihua XIao 4 (İD), Ayşegül tayLan ÖzKan 5 (İD)
Science of The Total Environment, 2021
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.