Comparison of RT-qPCR and Digital PCR Methods for Wastewater-Based Testing of SARS-CoV-2

Abstract

Wastewater-based epidemiology is an important tool for monitoring SARS-CoV-2 and other molecular targets in populations, using wastewater as a pooled sample. We compared the sensitivity, susceptibility to inhibition, and quantification of reverse transcription quantitative PCR (RT-qPCR), microfluidic well digital RT-PCR (RT-dPCR), and droplet digital RT-PCR (RT-ddPCR) measurements of SARS-CoV-2 (N1 gene target) and Pepper Mild Mottle Virus (PMMoV) RNA in 40 wastewater RNA extracts. All three methods were highly sensitive, but appeared less accurate at very low concentrations. Lower inhibition was observed for RT-ddPCR than RT-qPCR with both SARS-CoV-2 and PMMoV targets, but inhibition appeared to be mitigated by dilution of template RNA. The concentrations of N1 and PMMoV from all three methods were significantly correlated (Pearson's r=0.97-0.98 for N1 and r=0.89-0.93 for PMMoV), although RT-qPCR reported higher concentrations than digital methods. Taken together, this study provides support for the application of all three methods in wastewater-based epidemiology, with additional guidelines for the use of RT-qPCR.

Wastewater-based protocols for SARS-CoV-2: insights into virus concentration, extraction, and quantitation methods from two years of public health surveillance

“…6B). Several published studies corroborate our findings that RT-qPCR quantification may return higher concentrations of SARS-CoV-2 targets in comparison to the RT-dPCR in wastewater applications (e.g., Hinkle et al, 2022 57 ). The difference may be associated with circular plasmid standards overestimation (Hou et al 2010), 58 directly affecting the estimated N1 and N2 RT-qPCR concentrations.…”

“…23,60 Recent studies comparing the Qiacuity RT-dPCR platform and RT-qPCR confirm our results, indicating that the digital technology results in a significantly better signal to noise (lower LOD). 22,57 The published data on relative inhibition handling of the two platforms in wastewater application is more mixed. 62 More importantly, the universal adoption of dPCR platform could help with cross-laboratory comparisons of WBS data, given the inherent lack of a standard curve bias.…”

Wastewater-Based Epidemiology of SARS-CoV-2 and Other Respiratory Viruses: Bibliometric Tracking of the Last Decade and Emerging Research Directions

“…Another major advantage of digital PCR over its conventional counterpart is the direct absolute quantification of viral RNA due to its non-reliance on traditional standard curves. Additionally, due to the higher efficiency of digital PCR at lower concentrations of DNA/RNA and because of less interference from PCR inhibitors, digital PCR is reported to have lower limits of detection (LODs) and limits of quantification (LOQs) in comparison to qPCR, particularly in WBE studies [38,39]. This is quite an important aspect, as lower viral concentrations are usually expected in wastewater owing to high dilution [40].…”