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

Warish Ahmed, Stuart L. Simpson, Paul M. Bertsch, Kyle Bibby, Aaron Bivins, Linda L. Blackall, Sílvia Bofill-Mas, Albert Bosch, João Brandão, Phil M. Choi, Mark Ciesielski, Erica Donner, Nishita D'Souza, Andreas H. Farnleitner, Daniel Gerrity, Raul Gonzalez, John F. Griffith, Pradip Gyawali, Charles N. Haas, Kerry A. HamiltonHapuarachchige Chanditha Hapuarachchi, Valerie J. Harwood, Rehnuma Haque, Greg Jackson, Stuart J. Khan, Wesaal Khan, Masaaki Kitajima, Asja Korajkic, Giuseppina La Rosa, Blythe A. Layton, Erin Lipp, Sandra L. McLellan, Brian McMinn, Gertjan Medema, Suzanne Metcalfe, Wim G. Meijer, Jochen F. Mueller, Heather Murphy, Coleen C. Naughton, Rachel T. Noble, Sudhi Payyappat, Susan Petterson, Tarja Pitkänen, Veronica B. Rajal, Brandon Reyneke, Fernando A. Roman, Joan B. Rose, Marta Rusiñol, Michael J. Sadowsky, Laura Sala-Comorera, Yin Xiang Setoh, Samendra P. Sherchan, Kwanrawee Sirikanchana, Wendy Smith, Joshua A. Steele, Rosalie Subburg, Erin M. Symonds, Phong Thai, Kevin V. Thomas, Josh Tynan, Simon Toze, Janelle Thompson, Andy S. Whiteley, Judith Chui Ching Wong, Daisuke Sano, Stefan Wuertz, Irene Xagoraraki, Qian Zhang, Amity G. Zimmer-Faust, Orin C. Shanks

Research output: Contribution to journalReview articlepeer-review

134 Scopus citations


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.

Original languageEnglish (US)
Article number149877
JournalScience of the Total Environment
StatePublished - Jan 20 2022


  • COVID-19
  • False negative
  • False positive
  • RT-PCR
  • SARS-CoV-2
  • Surveillance
  • Wastewater

ASJC Scopus subject areas

  • Environmental Engineering
  • Environmental Chemistry
  • Waste Management and Disposal
  • Pollution


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