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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

Affiliations
Review

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

Aaron Bivins et al. Water Res. .

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.

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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 10th and 90th 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 99th 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.

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