Variability in RT-qPCR assay parameters indicates unreliable SARS-CoV-2 RNA quantification for wastewater surveillance

doi:10.1016/j.watres.2021.117516

Review

. 2021 Sep 15:203:117516.

doi: 10.1016/j.watres.2021.117516. Epub 2021 Aug 5.

Variability in RT-qPCR assay parameters indicates unreliable SARS-CoV-2 RNA quantification for wastewater surveillance

Aaron Bivins¹, Devrim Kaya², Kyle Bibby¹, Stuart L Simpson³, Stephen A Bustin⁴, Orin C Shanks⁵, Warish Ahmed⁶

Affiliations

¹ Department of Civil & Environmental Engineering & Earth Sciences, University of Notre Dame, Notre Dame, IN 46556, USA.
² School of Chemical, Biological, & Environmental Engineering, Oregon State University, Corvallis, OR 97331, USA.
³ CSIRO Land and Water, Lucas Heights, NSW 2234, Australia.
⁴ Medical Technology Research Center, Faculty of Health, Education and Social Care, Anglia Ruskin University, Chelmsford, Essex, CM1 1SQ, UK.
⁵ United States Environmental Protection Agency, Office of Research and Development, 26W Martin Luther King Jr. Drive, Cincinnati, OH, 45268, USA.
⁶ CSIRO Land and Water, Ecosciences Precinct, 41 Boggo Road, Dutton Park 4102, QLD, Australia. Electronic address: Warish.Ahmed@csiro.au.

PMID: 34412018
PMCID: PMC8341816
DOI: 10.1016/j.watres.2021.117516

Review

Variability in RT-qPCR assay parameters indicates unreliable SARS-CoV-2 RNA quantification for wastewater surveillance

Aaron Bivins et al. Water Res. 2021.

. 2021 Sep 15:203:117516.

doi: 10.1016/j.watres.2021.117516. Epub 2021 Aug 5.

Authors

Aaron Bivins¹, Devrim Kaya², Kyle Bibby¹, Stuart L Simpson³, Stephen A Bustin⁴, Orin C Shanks⁵, Warish Ahmed⁶

Affiliations

¹ Department of Civil & Environmental Engineering & Earth Sciences, University of Notre Dame, Notre Dame, IN 46556, USA.
² School of Chemical, Biological, & Environmental Engineering, Oregon State University, Corvallis, OR 97331, USA.
³ CSIRO Land and Water, Lucas Heights, NSW 2234, Australia.
⁴ Medical Technology Research Center, Faculty of Health, Education and Social Care, Anglia Ruskin University, Chelmsford, Essex, CM1 1SQ, UK.
⁵ United States Environmental Protection Agency, Office of Research and Development, 26W Martin Luther King Jr. Drive, Cincinnati, OH, 45268, USA.
⁶ CSIRO Land and Water, Ecosciences Precinct, 41 Boggo Road, Dutton Park 4102, QLD, Australia. Electronic address: Warish.Ahmed@csiro.au.

PMID: 34412018
PMCID: PMC8341816
DOI: 10.1016/j.watres.2021.117516

Abstract

Due to the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to generate large datasets aimed at detecting and quantifying SARS-CoV-2 RNA in wastewater. Although RT-qPCR is rapid and sensitive, there is no standard method yet, there are no certified quantification standards, and experiments are conducted using different assays, reagents, instruments, and data analysis protocols. These variations can induce errors in quantitative data reports, thereby potentially misleading interpretations, and conclusions. We review the SARS-CoV-2 wastewater surveillance literature focusing on variability of RT-qPCR data as revealed by inconsistent standard curves and associated parameters. We find that variation in these parameters and deviations from best practices, as described in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines suggest a frequent lack of reproducibility and reliability in quantitative measurements of SARS-CoV-2 RNA in wastewater.

Keywords: RT-qPCR; assay validity; quality assurance; quality control; standard curve; wastewater surveillance.

PubMed Disclaimer

Conflict of interest statement

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

Image, graphical abstract — **Graphical abstract**

Fig 1 — **Fig. 1**
SARS-CoV-2 US CDC N1 and N2 standard curve parameters, as reported in the literature: (A) y-intercept values, (B) slopes, (C) r² values, and (D) RT-qPCR efficiencies. The boxplots display the 10^th and 90^th percentiles (whiskers), interquartile range (box), median (line), and mean (+). The dashed line on panel (B) depicts an ideal slope of −3.32, on panel (C) depicts an r² value of 1.00, and on panel (D) depicts an ideal efficiency of 100%.

Fig 2 — **Fig. 2**
Dot plots of reported RT-qPCR standard curve parameters for CDC N1 and N2 assays: (A) y-intercepts, (B) slopes, (C) r² values, and (D) efficiencies stratified by linear synthetic RNA or cDNA versus plasmid control material. Individual values, mean, and standard deviation are displayed. The dashed line on panel (B) depicts an ideal slope of −3.32, on panel (C) depicts an r² value of 1.00, and on panel (D) depicts an ideal efficiency of 100%.

Fig 3 — **Fig. 3**
Log-linear regressions fit to the reported standard curves for CDC N1 assays (left; n = 22) and CDC N2 assays (right; *n =* 19) in the wastewater surveillance literature. Regressions fit to data from synthetic RNA or cDNA control materials are denoted in black, while those fit to plasmid control materials are denoted in pink. The shaded region displays the 99^th percentile for each. The slope (m), y-intercept (b), and r² values for the linear fit to each set of data are shown in the color corresponding to the regression line.

See this image and copyright information in PMC

Cited by

The effects of RT-qPCR standards on reproducibility and comparability in monitoring SARS-CoV-2 levels in wastewater.
Juutinen A, Tiwari A, Hokajärvi AM, Luomala O, Kolehmainen A, Nurmi E, Salmivirta E, Pitkänen T, Lipponen A. Juutinen A, et al. Sci Rep. 2024 Oct 26;14(1):25582. doi: 10.1038/s41598-024-77155-6. Sci Rep. 2024. PMID: 39462074 Free PMC article.
Inter-institutional laboratory standardization for SARS-CoV-2 surveillance through wastewater-based epidemiology applied to Mexico City.
Oyervides-Muñoz MA, Aguayo-Acosta A, de Los Cobos-Vasconcelos D, Carrillo-Reyes J, Espinosa-García AC, Campos E, Driver EM, Lucero-Saucedo SL, Armenta-Castro A, de la Rosa O, Martínez-Ruiz M, Barragán-Trinidad M, Vázquez-Salvador N, Silva-Magaña MA, Zavala-Méndez M, Iqbal HMN, Mazari-Hiriart M, Velazco H, Buitrón G, Noyola A, Halden RU, Sosa-Hernández JE, Parra-Saldívar R. Oyervides-Muñoz MA, et al. IJID Reg. 2024 Aug 21;12:100429. doi: 10.1016/j.ijregi.2024.100429. eCollection 2024 Sep. IJID Reg. 2024. PMID: 39318545 Free PMC article.
RT-qPCR Testing and Performance Metrics in the COVID-19 Era.
Bustin SA. Bustin SA. Int J Mol Sci. 2024 Aug 28;25(17):9326. doi: 10.3390/ijms25179326. Int J Mol Sci. 2024. PMID: 39273275 Free PMC article. Review.
Comparison of Different Reverse Transcriptase-Polymerase Chain Reaction-Based Methods for Wastewater Surveillance of SARS-CoV-2: Exploratory Study.
Länsivaara A, Lehto KM, Hyder R, Janhonen ES, Lipponen A, Heikinheimo A, Pitkänen T, Oikarinen S; WastPan Study Group. Länsivaara A, et al. JMIR Public Health Surveill. 2024 Aug 19;10:e53175. doi: 10.2196/53175. JMIR Public Health Surveill. 2024. PMID: 39158943 Free PMC article.
SARS-CoV-2 RNA Detection in Wastewater and Its Effective Correlation with Clinical Data during the Outbreak of COVID-19 in Salamanca.
Martínez de Alba ÁE, Morán-Diez ME, García-Prieto JC, García-Bernalt Diego J, Fernández-Soto P, Serrano León E, Monsalvo V, Casao M, Rubio MB, Hermosa R, Muro A, García-Roig M, Monte E. Martínez de Alba ÁE, et al. Int J Mol Sci. 2024 Jul 24;25(15):8071. doi: 10.3390/ijms25158071. Int J Mol Sci. 2024. PMID: 39125640 Free PMC article.

See all "Cited by" articles

References

1. Ahmed W., Tscharke B., Bertsch P.M., Bibby K., Bivins A., Choi P., Clarke L., Dwyer J., Edson J., Nguyen T.M.H., O’Brien J.W., Simpson S.L., Sherman P., Thomas K.V., Verhagen R., Zaugg J., Mueller J.F. SARS-CoV-2 RNA monitoring in wastewater as a potential early warning system for COVID-19 transmission in the community: A temporal case study. Sci. Total Environ. 2021;761 - 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., Simpson S., Bertsch P., Bibby K., Bivins A., Blackall L., Bofill-Mas S., Bosch A., Brandao J., Choi P., Ciesielski M., Donner E., D'Souza N., Farnleitner A., Gerrity D., Gonzalez R., Griffith J., Gyawali P., Haas C., Hamilton K., Hapuarachchi C., Harwood V., Haque R., Jackson G., Khan S., Khan W., Kitajima M., Korajkic A., La Rosa G., Layton B., Lipp E., McLellan S., McMinn B., Medema G., Metcalfe S., Meijer W., Mueller J., Murphy H., Naughton C., Noble R., Payyappat S., Petterson S., Pitkanen T., Rajal V., Reyneke B., Roman F., Rose J., Rusinol M., Sadowsky M., Sala-Comorera L., Setoh Y.X., Sherchan S., Sirikanchana K., Smith W., Steele J., Sabburg R., Symonds E., Thai P., Thomas K., Tynan J., Toze S., Thompson J., Whiteley A., Wong J., Sano D., Wuertz S., Xagoraraki I., Zhang Q., Zimmer-Faust A., Shanks O. Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance. Preprints. 2021 doi: 10.20944/preprints202104.0481.v1. 2021040481. - DOI - PMC - PubMed
1. Beinhauerova M., Babak V., Bertasi B., Boniotti M.B., Kralik P. Utilization of digital PCR in quantity verification of plasmid standards used in quantitative PCR. Front Mol Biosci. 2020;7:155. - PMC - PubMed
1. Bivins A., North D., Ahmad A., Ahmed W., Alm E., Been F., Bhattacharya P., Bijlsma L., Boehm A.B., Brown J., Buttiglieri G., Calabro V., Carducci A., Castiglioni S., Gurol Z.C., Chakraborty C., Costa F., Curcio S., de los reyes F.L., III, Delgado Vela J., Farkas K., Fernazdez-Casi X., Gerba C., Gerrity D., Girones R., Gonzzalez R., Haramoto E., Harris A., Holden P.A., Ispam M.T., Jones D.L., Kasprzyk-Hordern B., Kitajima M., Kotlarz N., Kumar M., Kuroda K., La Rosa G., Malpei F., Mautus M., Mclellan S.L., Medema G., Meschke J.S., Mueller J., Newton R.J., Noble R.T., van Nuijs A., Peccia J., Perkins T.A., Pickering A.J., Rose J., Sanchez G., Smith A., Stadler L., Stauber C., Thomas K., van der Voorn T., Wiggington K., Zhu K., Bibby K. Wastewater-based epidemiology: global collaborative to maximize contributions in the fight against COVID-19. Environ. Sci. Technol. 2020;54(13):7754–7757. - PubMed

Publication types

Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions

LinkOut - more resources

Full Text Sources
Medical
- MedlinePlus Health Information
Miscellaneous
- NCI CPTAC Assay Portal

[1] Ahmed W., Tscharke B., Bertsch P.M., Bibby K., Bivins A., Choi P., Clarke L., Dwyer J., Edson J., Nguyen T.M.H., O’Brien J.W., Simpson S.L., Sherman P., Thomas K.V., Verhagen R., Zaugg J., Mueller J.F. SARS-CoV-2 RNA monitoring in wastewater as a potential early warning system for COVID-19 transmission in the community: A temporal case study. Sci. Total Environ. 2021;761 - PMC - PubMed

[2] Ahmed W., Tscharke B., Bertsch P.M., Bibby K., Bivins A., Choi P., Clarke L., Dwyer J., Edson J., Nguyen T.M.H., O’Brien J.W., Simpson S.L., Sherman P., Thomas K.V., Verhagen R., Zaugg J., Mueller J.F. SARS-CoV-2 RNA monitoring in wastewater as a potential early warning system for COVID-19 transmission in the community: A temporal case study. Sci. Total Environ. 2021;761 - PMC - PubMed

[3] 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

[4] 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

[5] Ahmed W., Simpson S., Bertsch P., Bibby K., Bivins A., Blackall L., Bofill-Mas S., Bosch A., Brandao J., Choi P., Ciesielski M., Donner E., D'Souza N., Farnleitner A., Gerrity D., Gonzalez R., Griffith J., Gyawali P., Haas C., Hamilton K., Hapuarachchi C., Harwood V., Haque R., Jackson G., Khan S., Khan W., Kitajima M., Korajkic A., La Rosa G., Layton B., Lipp E., McLellan S., McMinn B., Medema G., Metcalfe S., Meijer W., Mueller J., Murphy H., Naughton C., Noble R., Payyappat S., Petterson S., Pitkanen T., Rajal V., Reyneke B., Roman F., Rose J., Rusinol M., Sadowsky M., Sala-Comorera L., Setoh Y.X., Sherchan S., Sirikanchana K., Smith W., Steele J., Sabburg R., Symonds E., Thai P., Thomas K., Tynan J., Toze S., Thompson J., Whiteley A., Wong J., Sano D., Wuertz S., Xagoraraki I., Zhang Q., Zimmer-Faust A., Shanks O. Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance. Preprints. 2021 doi: 10.20944/preprints202104.0481.v1. 2021040481. - DOI - PMC - PubMed

[6] Ahmed W., Simpson S., Bertsch P., Bibby K., Bivins A., Blackall L., Bofill-Mas S., Bosch A., Brandao J., Choi P., Ciesielski M., Donner E., D'Souza N., Farnleitner A., Gerrity D., Gonzalez R., Griffith J., Gyawali P., Haas C., Hamilton K., Hapuarachchi C., Harwood V., Haque R., Jackson G., Khan S., Khan W., Kitajima M., Korajkic A., La Rosa G., Layton B., Lipp E., McLellan S., McMinn B., Medema G., Metcalfe S., Meijer W., Mueller J., Murphy H., Naughton C., Noble R., Payyappat S., Petterson S., Pitkanen T., Rajal V., Reyneke B., Roman F., Rose J., Rusinol M., Sadowsky M., Sala-Comorera L., Setoh Y.X., Sherchan S., Sirikanchana K., Smith W., Steele J., Sabburg R., Symonds E., Thai P., Thomas K., Tynan J., Toze S., Thompson J., Whiteley A., Wong J., Sano D., Wuertz S., Xagoraraki I., Zhang Q., Zimmer-Faust A., Shanks O. Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance. Preprints. 2021 doi: 10.20944/preprints202104.0481.v1. 2021040481. - DOI - PMC - PubMed

[7] Beinhauerova M., Babak V., Bertasi B., Boniotti M.B., Kralik P. Utilization of digital PCR in quantity verification of plasmid standards used in quantitative PCR. Front Mol Biosci. 2020;7:155. - PMC - PubMed

[8] Beinhauerova M., Babak V., Bertasi B., Boniotti M.B., Kralik P. Utilization of digital PCR in quantity verification of plasmid standards used in quantitative PCR. Front Mol Biosci. 2020;7:155. - PMC - PubMed

[9] Bivins A., North D., Ahmad A., Ahmed W., Alm E., Been F., Bhattacharya P., Bijlsma L., Boehm A.B., Brown J., Buttiglieri G., Calabro V., Carducci A., Castiglioni S., Gurol Z.C., Chakraborty C., Costa F., Curcio S., de los reyes F.L., III, Delgado Vela J., Farkas K., Fernazdez-Casi X., Gerba C., Gerrity D., Girones R., Gonzzalez R., Haramoto E., Harris A., Holden P.A., Ispam M.T., Jones D.L., Kasprzyk-Hordern B., Kitajima M., Kotlarz N., Kumar M., Kuroda K., La Rosa G., Malpei F., Mautus M., Mclellan S.L., Medema G., Meschke J.S., Mueller J., Newton R.J., Noble R.T., van Nuijs A., Peccia J., Perkins T.A., Pickering A.J., Rose J., Sanchez G., Smith A., Stadler L., Stauber C., Thomas K., van der Voorn T., Wiggington K., Zhu K., Bibby K. Wastewater-based epidemiology: global collaborative to maximize contributions in the fight against COVID-19. Environ. Sci. Technol. 2020;54(13):7754–7757. - PubMed

[10] Bivins A., North D., Ahmad A., Ahmed W., Alm E., Been F., Bhattacharya P., Bijlsma L., Boehm A.B., Brown J., Buttiglieri G., Calabro V., Carducci A., Castiglioni S., Gurol Z.C., Chakraborty C., Costa F., Curcio S., de los reyes F.L., III, Delgado Vela J., Farkas K., Fernazdez-Casi X., Gerba C., Gerrity D., Girones R., Gonzzalez R., Haramoto E., Harris A., Holden P.A., Ispam M.T., Jones D.L., Kasprzyk-Hordern B., Kitajima M., Kotlarz N., Kumar M., Kuroda K., La Rosa G., Malpei F., Mautus M., Mclellan S.L., Medema G., Meschke J.S., Mueller J., Newton R.J., Noble R.T., van Nuijs A., Peccia J., Perkins T.A., Pickering A.J., Rose J., Sanchez G., Smith A., Stadler L., Stauber C., Thomas K., van der Voorn T., Wiggington K., Zhu K., Bibby K. Wastewater-based epidemiology: global collaborative to maximize contributions in the fight against COVID-19. Environ. Sci. Technol. 2020;54(13):7754–7757. - PubMed

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Variability in RT-qPCR assay parameters indicates unreliable SARS-CoV-2 RNA quantification for wastewater surveillance

Affiliations

Variability in RT-qPCR assay parameters indicates unreliable SARS-CoV-2 RNA quantification for wastewater surveillance

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

LinkOut - more resources

Full Text Sources

Medical

Miscellaneous

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Related information

LinkOut - more resources

Full Text Sources

Medical

Miscellaneous