Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance

doi:10.1016/j.scitotenv.2021.149877

Review

. 2022 Jan 20:805:149877.

doi: 10.1016/j.scitotenv.2021.149877. Epub 2021 Aug 25.

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance

Warish Ahmed¹, Stuart L Simpson², Paul M Bertsch³, Kyle Bibby⁴, Aaron Bivins⁴, Linda L Blackall⁵, Sílvia Bofill-Mas⁶, Albert Bosch⁷, João Brandão⁸, Phil M Choi⁹, Mark Ciesielski¹⁰, Erica Donner¹¹, Nishita D'Souza¹², Andreas H Farnleitner¹³, Daniel Gerrity¹⁴, Raul Gonzalez¹⁵, John F Griffith¹⁶, Pradip Gyawali¹⁷, Charles N Haas¹⁸, Kerry A Hamilton¹⁹, Hapuarachchige Chanditha Hapuarachchi²⁰, Valerie J Harwood²¹, Rehnuma Haque²², Greg Jackson²³, Stuart J Khan²⁴, Wesaal Khan²⁵, Masaaki Kitajima²⁶, Asja Korajkic²⁷, Giuseppina La Rosa²⁸, Blythe A Layton²⁹, Erin Lipp³⁰, Sandra L McLellan³¹, Brian McMinn²⁷, Gertjan Medema³², Suzanne Metcalfe³, Wim G Meijer³³, Jochen F Mueller³⁴, Heather Murphy³⁵, Coleen C Naughton³⁶, Rachel T Noble¹⁰, Sudhi Payyappat³⁷, Susan Petterson³⁸, Tarja Pitkänen³⁹, Veronica B Rajal⁴⁰, Brandon Reyneke²⁵, Fernando A Roman Jr³⁶, Joan B Rose¹², Marta Rusiñol⁴¹, Michael J Sadowsky⁴², Laura Sala-Comorera³³, Yin Xiang Setoh²⁰, Samendra P Sherchan⁴³, Kwanrawee Sirikanchana⁴⁴, Wendy Smith³, Joshua A Steele¹⁶, Rosalie Sabburg⁴⁵, Erin M Symonds⁴⁶, Phong Thai³⁴, Kevin V Thomas³⁴, Josh Tynan³⁴, Simon Toze³, Janelle Thompson⁴⁷, Andy S Whiteley⁴⁸, Judith Chui Ching Wong²⁰, Daisuke Sano⁴⁹, Stefan Wuertz⁵⁰, Irene Xagoraraki⁵¹, Qian Zhang⁴², Amity G Zimmer-Faust¹⁶, Orin C Shanks²⁷

Affiliations

¹ CSIRO Land and Water, Ecosciences Precinct, 41 Boggo Road, QLD 4102, Australia. Electronic address: Warish.Ahmed@csiro.au.
² CSIRO Land and Water, Lucas Heights, NSW 2234, Australia.
³ CSIRO Land and Water, Ecosciences Precinct, 41 Boggo Road, QLD 4102, Australia.
⁴ Department of Civil & Environmental Engineering & Earth Science, University of Notre Dame, 156 Fitzpatrick Hall, Notre Dame, IN 46556, USA.
⁵ School of BioSciences, The University of Melbourne, Melbourne, VIC, Australia.
⁶ Laboratory of Virus Contaminants of Water and Food, Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain.
⁷ Enteric Virus Laboratory, Department of Genetics, Microbiology and Statistics, University of Barcelona, Avda. Diagonal 643, 08028 Barcelona, Spain.
⁸ Department of Environmental Health, National Institute of Health Dr. Ricardo Jorge, Lisboa, Portugal.
⁹ Water Unit, Health Protection Branch, Prevention Division, Queensland Health, QLD, Australia; The University of Queensland, Queensland Alliance for Environmental Health Sciences, QLD, Australia.
¹⁰ University of North Carolina at Chapel Hill, Institute of Marine Sciences, Morehead City, NC, United States.
¹¹ Future Industries Institute, University of South Australia, University Boulevard, Mawson Lakes, SA 5095, Australia.
¹² Department of Fisheries and Wildlife, Michigan State University, E. Lansing, MI, USA.
¹³ Institute of Chemical, Environmental & Bioscience Engineering, Research Group Environmental Microbiology and Molecular Diagnostic, 166/5/3, Technische Universität Wien, Vienna, Austria; Research Division Water Quality and Health, Department Pharmacology, Physiology and Microbiology, Karl Landsteiner University of Health Sciences, Dr. Karl-Dorrek-Straβe 30, 3500 Krems an der Donau, Austria.
¹⁴ Southern Nevada Water Authority, P.O. Box 99954, Las Vegas, NV 89193, USA.
¹⁵ Hampton Roads Sanitation District, 1434 Air Rail Avenue, Virginia Beach, VA 23455, USA.
¹⁶ Southern California Coastal Water Research Project, Costa Mesa, CA 92626, USA.
¹⁷ Institute of Environmental Science and Research Ltd (ESR), Porirua 5240, New Zealand.
¹⁸ Drexel University, Philadelphia, PA, USA.
¹⁹ School of Sustainable Engineering and the Built Environment and The Biodesign Institute Center for Environmental Health Engineering, Arizona State University, Tempe, AZ 85287, USA.
²⁰ Environmental Health Institute, National Environment Agency, Singapore.
²¹ Department of Integrative Biology, University of South Florida, Tampa, FL, USA.
²² Environmental Interventions Unit, Icddr,b, 68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh.
²³ Water Unit, Health Protection Branch, Prevention Division, Queensland Health, QLD, Australia.
²⁴ Water Research Centre, School of Civil and Environmental Engineering, University of New South Wales, NSW 2052, Australia.
²⁵ Department of Microbiology, Faculty of Science, Stellenbosch University, Private Bag X1, Stellenbosch 7602, South Africa.
²⁶ Division of Environmental Engineering, Faculty of Engineering, Hokkaido University, North 13 West 8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan.
²⁷ United States Environmental Protection Agency, Office of Research and Development, 26W Martin Luther King Jr. Drive, Cincinnati, OH 45268, USA.
²⁸ Department of Environment and Health, Istituto Superiore di Sanità, Rome, Italy.
²⁹ Department of Research & Innovation, Clean Water Services, Hillsboro, OR, USA.
³⁰ Environmental Health Sciences Department, University of Georgia, Athens, GA 30602, USA.
³¹ School of Freshwater Sciences, University of Wisconsin-Milwaukee, WI, USA.
³² KWR Water Research Institute, Groningenhaven 7, 3433 PE Nieuwegein, the Netherlands.
³³ UCD School of Biomolecular and Biomedical Science, University College Dublin, Dublin, Ireland.
³⁴ The University of Queensland, Queensland Alliance for Environmental Health Sciences, QLD, Australia.
³⁵ Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada.
³⁶ University of California Merced, Department of Civil and Environmental Engineering, 5200 N. Lake Rd., Merced, CA 95343, USA.
³⁷ Sydney Water, 1 Smith Street, Parramatta, NSW 2150, Australia.
³⁸ Water and Health Pty Ltd., 13 Lord St, North Sydney, NSW 2060, Australia; School of Medicine, Griffith University, Parklands Drive, Gold Coast, Australia.
³⁹ Finnish Institute for Health and Welfare, Expert Microbiology Unit, P.O. Box 95, FI-70701 Kuopio, Finland; University of Helsinki, Faculty of Veterinary Medicine, Department of Food Hygiene and Environmental Health, P.O. Box 66, FI-00014, Finland.
⁴⁰ Facultad de Ingeniería and Instituto de Investigaciones para la Industria Química (INIQUI) - CONICET and Universidad Nacional de Salta, Av. Bolivia 5150, Salta, Argentina.
⁴¹ Institute of Environmental Assessment & Water Research (IDAEA), CSIC, Barcelona, Spain.
⁴² Biotechnology Institute and Department of Soil, Water, and Climate, University of Minnesota, St. Paul, MN, USA.
⁴³ Department of Environmental Health Sciences, Tulane University, 1440 Canal Street, New Orleans, LA 70112, USA.
⁴⁴ Research Laboratory of Biotechnology, Chulabhorn Research Institute, 54 Kampangpetch 6 Road, Laksi, Bangkok 10210, Thailand.
⁴⁵ CSIRO Agriculture and Food, Bioscience Precinct, St Lucia, QLD 4067, Australia.
⁴⁶ College of Marine Science, University of South Florida, St. Petersburg, FL, USA.
⁴⁷ Asian School of the Environment, Nanyang Technological University, Singapore 639798, Singapore; Singapore Centre for Environmental Life Sciences Engineering (SCELSE) Singapore 637551.
⁴⁸ CSIRO Land and Water, Floreat, WA 6012, Australia.
⁴⁹ Department of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, Aoba 6-6-06, Aramaki, Aoba-Ku, Sendai, Miyagi 980-8597, Japan.
⁵⁰ Singapore Centre for Environmental Life Sciences Engineering (SCELSE) Singapore 637551; School of Civil and Environmental Engineering, Nanyang Technological University, Singapore 639798.
⁵¹ Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, USA.

PMID: 34818780
PMCID: PMC8386095
DOI: 10.1016/j.scitotenv.2021.149877

Review

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance

Warish Ahmed et al. Sci Total Environ. 2022.

. 2022 Jan 20:805:149877.

doi: 10.1016/j.scitotenv.2021.149877. Epub 2021 Aug 25.

Authors

Affiliations

¹ CSIRO Land and Water, Ecosciences Precinct, 41 Boggo Road, QLD 4102, Australia. Electronic address: Warish.Ahmed@csiro.au.
² CSIRO Land and Water, Lucas Heights, NSW 2234, Australia.
³ CSIRO Land and Water, Ecosciences Precinct, 41 Boggo Road, QLD 4102, Australia.
⁴ Department of Civil & Environmental Engineering & Earth Science, University of Notre Dame, 156 Fitzpatrick Hall, Notre Dame, IN 46556, USA.
⁵ School of BioSciences, The University of Melbourne, Melbourne, VIC, Australia.
⁶ Laboratory of Virus Contaminants of Water and Food, Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain.
⁷ Enteric Virus Laboratory, Department of Genetics, Microbiology and Statistics, University of Barcelona, Avda. Diagonal 643, 08028 Barcelona, Spain.
⁸ Department of Environmental Health, National Institute of Health Dr. Ricardo Jorge, Lisboa, Portugal.
⁹ Water Unit, Health Protection Branch, Prevention Division, Queensland Health, QLD, Australia; The University of Queensland, Queensland Alliance for Environmental Health Sciences, QLD, Australia.
¹⁰ University of North Carolina at Chapel Hill, Institute of Marine Sciences, Morehead City, NC, United States.
¹¹ Future Industries Institute, University of South Australia, University Boulevard, Mawson Lakes, SA 5095, Australia.
¹² Department of Fisheries and Wildlife, Michigan State University, E. Lansing, MI, USA.
¹³ Institute of Chemical, Environmental & Bioscience Engineering, Research Group Environmental Microbiology and Molecular Diagnostic, 166/5/3, Technische Universität Wien, Vienna, Austria; Research Division Water Quality and Health, Department Pharmacology, Physiology and Microbiology, Karl Landsteiner University of Health Sciences, Dr. Karl-Dorrek-Straβe 30, 3500 Krems an der Donau, Austria.
¹⁴ Southern Nevada Water Authority, P.O. Box 99954, Las Vegas, NV 89193, USA.
¹⁵ Hampton Roads Sanitation District, 1434 Air Rail Avenue, Virginia Beach, VA 23455, USA.
¹⁶ Southern California Coastal Water Research Project, Costa Mesa, CA 92626, USA.
¹⁷ Institute of Environmental Science and Research Ltd (ESR), Porirua 5240, New Zealand.
¹⁸ Drexel University, Philadelphia, PA, USA.
¹⁹ School of Sustainable Engineering and the Built Environment and The Biodesign Institute Center for Environmental Health Engineering, Arizona State University, Tempe, AZ 85287, USA.
²⁰ Environmental Health Institute, National Environment Agency, Singapore.
²¹ Department of Integrative Biology, University of South Florida, Tampa, FL, USA.
²² Environmental Interventions Unit, Icddr,b, 68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh.
²³ Water Unit, Health Protection Branch, Prevention Division, Queensland Health, QLD, Australia.
²⁴ Water Research Centre, School of Civil and Environmental Engineering, University of New South Wales, NSW 2052, Australia.
²⁵ Department of Microbiology, Faculty of Science, Stellenbosch University, Private Bag X1, Stellenbosch 7602, South Africa.
²⁶ Division of Environmental Engineering, Faculty of Engineering, Hokkaido University, North 13 West 8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan.
²⁷ United States Environmental Protection Agency, Office of Research and Development, 26W Martin Luther King Jr. Drive, Cincinnati, OH 45268, USA.
²⁸ Department of Environment and Health, Istituto Superiore di Sanità, Rome, Italy.
²⁹ Department of Research & Innovation, Clean Water Services, Hillsboro, OR, USA.
³⁰ Environmental Health Sciences Department, University of Georgia, Athens, GA 30602, USA.
³¹ School of Freshwater Sciences, University of Wisconsin-Milwaukee, WI, USA.
³² KWR Water Research Institute, Groningenhaven 7, 3433 PE Nieuwegein, the Netherlands.
³³ UCD School of Biomolecular and Biomedical Science, University College Dublin, Dublin, Ireland.
³⁴ The University of Queensland, Queensland Alliance for Environmental Health Sciences, QLD, Australia.
³⁵ Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada.
³⁶ University of California Merced, Department of Civil and Environmental Engineering, 5200 N. Lake Rd., Merced, CA 95343, USA.
³⁷ Sydney Water, 1 Smith Street, Parramatta, NSW 2150, Australia.
³⁸ Water and Health Pty Ltd., 13 Lord St, North Sydney, NSW 2060, Australia; School of Medicine, Griffith University, Parklands Drive, Gold Coast, Australia.
³⁹ Finnish Institute for Health and Welfare, Expert Microbiology Unit, P.O. Box 95, FI-70701 Kuopio, Finland; University of Helsinki, Faculty of Veterinary Medicine, Department of Food Hygiene and Environmental Health, P.O. Box 66, FI-00014, Finland.
⁴⁰ Facultad de Ingeniería and Instituto de Investigaciones para la Industria Química (INIQUI) - CONICET and Universidad Nacional de Salta, Av. Bolivia 5150, Salta, Argentina.
⁴¹ Institute of Environmental Assessment & Water Research (IDAEA), CSIC, Barcelona, Spain.
⁴² Biotechnology Institute and Department of Soil, Water, and Climate, University of Minnesota, St. Paul, MN, USA.
⁴³ Department of Environmental Health Sciences, Tulane University, 1440 Canal Street, New Orleans, LA 70112, USA.
⁴⁴ Research Laboratory of Biotechnology, Chulabhorn Research Institute, 54 Kampangpetch 6 Road, Laksi, Bangkok 10210, Thailand.
⁴⁵ CSIRO Agriculture and Food, Bioscience Precinct, St Lucia, QLD 4067, Australia.
⁴⁶ College of Marine Science, University of South Florida, St. Petersburg, FL, USA.
⁴⁷ Asian School of the Environment, Nanyang Technological University, Singapore 639798, Singapore; Singapore Centre for Environmental Life Sciences Engineering (SCELSE) Singapore 637551.
⁴⁸ CSIRO Land and Water, Floreat, WA 6012, Australia.
⁴⁹ Department of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, Aoba 6-6-06, Aramaki, Aoba-Ku, Sendai, Miyagi 980-8597, Japan.
⁵⁰ Singapore Centre for Environmental Life Sciences Engineering (SCELSE) Singapore 637551; School of Civil and Environmental Engineering, Nanyang Technological University, Singapore 639798.
⁵¹ Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, USA.

PMID: 34818780
PMCID: PMC8386095
DOI: 10.1016/j.scitotenv.2021.149877

Abstract

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.

Keywords: COVID-19; False negative; False positive; RT-PCR; SARS-CoV-2; Surveillance; Wastewater.

PubMed Disclaimer

Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Unlabelled Image — **Graphical abstract**

**Fig. 1**
A conceptual diagram of a typical sewer catchment with sampling points and analytical workflow of SARS-CoV-2 RNA surveillance in wastewater; MH: Maintenance hole.

**Fig. 2**
Under low-prevalence conditions, some random sewage volumes (orange ellipses and circles) contain detectable SARS-CoV-2 RNA, while others (brown matrix) do not.

**Fig. 3**
a: Probability of a single small grab sample (relative to SARS-CoV-2 dispersion volume) being negative, assuming a sewer catchment population of 100,000 people, an average dry weather flow of 0.2 m³/person/day, and dispersion volumes of 1 m³/day, 10 m³/day, or 100 m³/day. 3b: Probability of all samples being negative for n = 1, 10, 100, or 1000 samples.

**Fig. 4**
SARS-CoV-2 concentrations measured by RT-PCR in wastewater samples that were untreated (gray bars) or pasteurized (salmon bars). The mean concentration in pasteurized samples was censored below the detection limit; values that were detected but not quantifiable (DNQ) were replaced with half the limit of quantification for statistical analysis. Samples in which the target was not detected (ND) were assigned a value of 1.0. Data were obtained partially from Steele et al. (2021).

**Fig. 5**
Recovery (mean ± SD) of seeded SARS-CoV-2 RNA from 10 wastewater treatment plants (WWTP A-J) using a concentrating pipette Select™ (CP Select™). Each WWTP was sampled three times. Data were obtained partially from Ahmed et al. (2021).

**Fig. 6**
Estimated method limit of detection (MLOD) according to the final volume of concentrated samples. The MLOD was calculated assuming 323 GC as the minimum number detected for a single RT-qPCR reaction using CDC N1 assay and a mean volume of viral concentrate of 300 μL for the CP Select™, 500 μL for SMF and 240 μL for Centricon®. A 100% recovery was assumed for the entire concentration, RNA extraction and detection process. SMF: skimmed milk flocculation. Data obtained partially from Forés et al. (2021).

**Fig. 7**
Quantitation cycle (Cq) values of the US CDC N1 and N2 RT-PCR assays determined using the Australian National Measurement Institute (NMI) SARS-CoV-2 calibrant comprising six gravimetric dilutions at 600, 245, 60, 18.5, 6.5, and 2 GC/5 μL of RNA. The Bio-Rad CFX96 platform was used for the RT-PCR amplification of the N1 and N2 assays.

**Fig. 8**
SARS-CoV-2 (CDC N2) concentrations over time in gene copies (GC)/mL from seven distinct wastewater treatment plants (WWTPs) servicing in south–east Virginia. The WWTPs serve catchment populations ranging from 69,509 to 343,016 individuals. Weekly sampling events began May 14, 2020 and continued through July 14, 2020. WWTPs are identified first by the arbitrary facility number, followed by the corresponding sample collection event (e.g., WWTP-5-3 would refer to the third collection event for WWTP 5). Data was partially obtained from Ciesielski et al. (2021).

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References

1. Ahmed W., Harwood V.J., Gyawali P., Sidhu J.P.S., Toze S. Comparison of concentration methods for quantitative detection of sewage-associated viral markers in environmental waters. Appl. Environ. Microbiol. 2015;81(6):2042–2049. - PMC - PubMed
1. Ahmed W., Angel N., Edson J., Bibby K., Bivins A., Brien J.W.O., Choi P.M., Kitajima M., Simpson S.L., Li J., Tscharke B., Verhagen R., Smith W.J.M., Zaugg J., Dierens L., Hugenholtz P., Thomas K.V., Mueller J.F. First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: a proof of concept for the wastewater surveillance of COVID-19 in the community. Sci. Total Environ. 2020;728 - PMC - PubMed
1. Ahmed W., Bertsch P.M., Bibby K., Haramoto E., Hewitt J., Huygens F., Gyawali P., Korajkic A., Riddell S., Sherchan S.P., Simpson S.L., Sirikanchana K., Symonds E.M., Verhagen R., Vasan S.S., Kitajima M., Bivins A. Decay of SARS-CoV-2 and surrogate murine hepatitis virus RNA in untreated wastewater to inform application in wastewater-based epidemiology. Environ. Res. 2020;191 - PMC - PubMed
1. Ahmed W., Bivins A., Bertsch P.M., Bibby K., Choi P.M., Farkas K., Gyawali P., Hamilton K.A., Haramoto E., Kitajima M., Simpson S.L., Tandukar S., Thomas K.V., Mueller J.F. Surveillance of SARS-CoV-2 RNA in wastewater: methods optimization and quality control are crucial for generating reliable public health information. Curr. Opin. Environ. Sci. Health. 2020;17:82–93. - PMC - PubMed
1. Ahmed W., Bertsch P.M., Bivins A., Bibby K., Farkas K., Gathercole A., Haramoto E., Gyawali P., Korajkic A., McMinn B.R., Mueller J.F., Simpson S.L., Smith W.J.M., Symonds E.M., Thomas K.V., Verhagen R., Kitajima M. Comparison of virus concentration methods for the RT-qPCR based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater. Sci. Total Environ. 2020;739 - PMC - PubMed

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[1] Ahmed W., Harwood V.J., Gyawali P., Sidhu J.P.S., Toze S. Comparison of concentration methods for quantitative detection of sewage-associated viral markers in environmental waters. Appl. Environ. Microbiol. 2015;81(6):2042–2049. - PMC - PubMed

[2] Ahmed W., Harwood V.J., Gyawali P., Sidhu J.P.S., Toze S. Comparison of concentration methods for quantitative detection of sewage-associated viral markers in environmental waters. Appl. Environ. Microbiol. 2015;81(6):2042–2049. - PMC - PubMed

[3] Ahmed W., Angel N., Edson J., Bibby K., Bivins A., Brien J.W.O., Choi P.M., Kitajima M., Simpson S.L., Li J., Tscharke B., Verhagen R., Smith W.J.M., Zaugg J., Dierens L., Hugenholtz P., Thomas K.V., Mueller J.F. First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: a proof of concept for the wastewater surveillance of COVID-19 in the community. Sci. Total Environ. 2020;728 - PMC - PubMed

[4] Ahmed W., Angel N., Edson J., Bibby K., Bivins A., Brien J.W.O., Choi P.M., Kitajima M., Simpson S.L., Li J., Tscharke B., Verhagen R., Smith W.J.M., Zaugg J., Dierens L., Hugenholtz P., Thomas K.V., Mueller J.F. First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: a proof of concept for the wastewater surveillance of COVID-19 in the community. Sci. Total Environ. 2020;728 - PMC - PubMed

[5] Ahmed W., Bertsch P.M., Bibby K., Haramoto E., Hewitt J., Huygens F., Gyawali P., Korajkic A., Riddell S., Sherchan S.P., Simpson S.L., Sirikanchana K., Symonds E.M., Verhagen R., Vasan S.S., Kitajima M., Bivins A. Decay of SARS-CoV-2 and surrogate murine hepatitis virus RNA in untreated wastewater to inform application in wastewater-based epidemiology. Environ. Res. 2020;191 - PMC - PubMed

[6] Ahmed W., Bertsch P.M., Bibby K., Haramoto E., Hewitt J., Huygens F., Gyawali P., Korajkic A., Riddell S., Sherchan S.P., Simpson S.L., Sirikanchana K., Symonds E.M., Verhagen R., Vasan S.S., Kitajima M., Bivins A. Decay of SARS-CoV-2 and surrogate murine hepatitis virus RNA in untreated wastewater to inform application in wastewater-based epidemiology. Environ. Res. 2020;191 - PMC - PubMed

[7] Ahmed W., Bivins A., Bertsch P.M., Bibby K., Choi P.M., Farkas K., Gyawali P., Hamilton K.A., Haramoto E., Kitajima M., Simpson S.L., Tandukar S., Thomas K.V., Mueller J.F. Surveillance of SARS-CoV-2 RNA in wastewater: methods optimization and quality control are crucial for generating reliable public health information. Curr. Opin. Environ. Sci. Health. 2020;17:82–93. - PMC - PubMed

[8] Ahmed W., Bivins A., Bertsch P.M., Bibby K., Choi P.M., Farkas K., Gyawali P., Hamilton K.A., Haramoto E., Kitajima M., Simpson S.L., Tandukar S., Thomas K.V., Mueller J.F. Surveillance of SARS-CoV-2 RNA in wastewater: methods optimization and quality control are crucial for generating reliable public health information. Curr. Opin. Environ. Sci. Health. 2020;17:82–93. - PMC - PubMed

[9] Ahmed W., Bertsch P.M., Bivins A., Bibby K., Farkas K., Gathercole A., Haramoto E., Gyawali P., Korajkic A., McMinn B.R., Mueller J.F., Simpson S.L., Smith W.J.M., Symonds E.M., Thomas K.V., Verhagen R., Kitajima M. Comparison of virus concentration methods for the RT-qPCR based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater. Sci. Total Environ. 2020;739 - PMC - PubMed

[10] Ahmed W., Bertsch P.M., Bivins A., Bibby K., Farkas K., Gathercole A., Haramoto E., Gyawali P., Korajkic A., McMinn B.R., Mueller J.F., Simpson S.L., Smith W.J.M., Symonds E.M., Thomas K.V., Verhagen R., Kitajima M. Comparison of virus concentration methods for the RT-qPCR based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater. Sci. Total Environ. 2020;739 - PMC - PubMed

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Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance

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Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance

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Abstract

Conflict of interest statement

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