RT-qPCR quantification of SARS-CoV-2 in wastewater: An in-depth investigation of standards
Natacha Prostran (1), Robert Delatolla (2), Richard Goulet (1)
(1) Department of Earth and Environmental Sciences, University of Ottawa
(2) Department of Civil/Environmental Engineering, University of Ottawa
Natacha Prostran (1), Robert Delatolla (2), Richard Goulet (1)
(1) Department of Earth and Environmental Sciences, University of Ottawa
(2) Department of Civil/Environmental Engineering, University of Ottawa
The current global pandemic that ensued upon the arrival of the SARS-CoV-2 coronavirus has placed an enormous burden on the health care system. The need for more accurate and reliable tracking of Covid-19 incidence has only become more apparent as positive cases continue to rise and new SARS-CoV-2 variants are introduced. Aside from clinical testing, researchers and municipalities have turned to wastewater-based epidemiology (WBE) as a means to accurately quantify true positive cases within a population. One of the main methods used to quantify viral signal is through RT-qPCR in which a standard curve is used to directly quantify the number of viral copies in a sample. However, the standards used to construct these standard curves are inconsistent between research groups, putting into question the reliability of the results. The standards that are currently being used are made up of either synthetic RNA/DNA, Plasmid DNA, cDNA, gBlock fragments, or extracted RNA. Varying standards generate inconsistent standard curves, ultimately leading to a possible over or underestimation of viral signal. An in-depth literature review was conducted to investigate the types of standards being used and the prevalence of each type. The review not only looks at wastewater-related studies, but others within the clinical and public health fields as well. In addition, an experimental component to the research project evaluates the performance of 7 commercially available standards during RT-qPCR amplification and how accurately each standard is able to quantify viral signal. Results showed that the two most commonly used standards were synthetic RNA and plasmid DNA, which are exceptionally different from one another in their structure, stability, and behaviour during RT-qPCR. Linearized plasmid quantified the lowest viral signal while some synthetic RNAs were more accurate, and others caused overestimations. The aim of this research is to highlight the importance of choosing the right template material (DNA or RNA) for the construction of a standard curve. While plasmid DNA is more stable, easy to prepare, and able to be stored for longer periods of time than RNA, it does not account for the variability in the reverse transcription (RT) step of RT-qPCR. For this reason, a synthetic RNA standard is recommended as it incorporates the RT step and closely resembles the genetic material of SARS-CoV-2, the target being quantified. Still, not all synthetic RNAs provide optimal quantification, therefore careful consideration of RNAs available must be had before choosing the appropriate standard.