Preprint Review Version 1 Preserved in Portico This version is not peer-reviewed

Inherent Bias of SARS-CoV-2 RNA Quantification for Wastewater Surveillance Due to Variable RT-qPCR Assay Parameters

Version 1 : Received: 9 June 2021 / Approved: 11 June 2021 / Online: 11 June 2021 (14:10:51 CEST)

How to cite: Bivins, A.; Kaya, D.; Bibby, K.; Simpson, S.; Bustin, S.; Shanks, O.; Ahmed, W. Inherent Bias of SARS-CoV-2 RNA Quantification for Wastewater Surveillance Due to Variable RT-qPCR Assay Parameters. Preprints 2021, 2021060320 (doi: 10.20944/preprints202106.0320.v1). Bivins, A.; Kaya, D.; Bibby, K.; Simpson, S.; Bustin, S.; Shanks, O.; Ahmed, W. Inherent Bias of SARS-CoV-2 RNA Quantification for Wastewater Surveillance Due to Variable RT-qPCR Assay Parameters. Preprints 2021, 2021060320 (doi: 10.20944/preprints202106.0320.v1).

Abstract

The coronavirus disease 2019 (COVID-19) pandemic has led to wastewater surveillance becoming an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. As a result, molecular methods, in particular reverse transcription-quantitative PCR (RT-qPCR), have been employed to generate large data sets aimed at the detection and quantification of SARS-CoV-2 in wastewater. Although RT-qPCR is rapid and sensitive, there is no standard method that fits all use cases, there are no certified quantification standards and experiments are carried out using numerous different assays, reagents, instruments, and data analysis protocols. These variations can lead to the reporting of erroneous quantitative data resulting in potentially misleading interpretations and conclusions. We have reviewed the SARS-CoV-2 wastewater surveillance literature focusing on the 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 lack of reproducibility and reliability in quantitative measurements of SARS-CoV-2 RNA in wastewater.

Subject Areas

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

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