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. 2022 Nov 11;2(11):1871-1880.
doi: 10.1021/acsestwater.1c00387. Epub 2022 Jan 28.

Comparison of RT-qPCR and RT-dPCR Platforms for the Trace Detection of SARS-CoV-2 RNA in Wastewater

Warish Ahmed  1 , Wendy J M Smith  1 , Suzanne Metcalfe  1 , Greg Jackson  2 , Phil M Choi  2 , Mary Morrison  2 , Daniel Field  2 , Pradip Gyawali  3 , Aaron Bivins  4 , Kyle Bibby  4 , Stuart L Simpson  5
Affiliations

Comparison of RT-qPCR and RT-dPCR Platforms for the Trace Detection of SARS-CoV-2 RNA in Wastewater

Warish Ahmed et al. ACS ES T Water. .

Abstract

We compared reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RT digital PCR (RT-dPCR) platforms for the trace detection of SARS-CoV-2 RNA in low-prevalence COVID-19 locations in Queensland, Australia, using CDC N1 and CDC N2 assays. The assay limit of detection (ALOD), PCR inhibition rates, and performance characteristics of each assay, along with the positivity rates with the RT-qPCR and RT-dPCR platforms, were evaluated by seeding known concentrations of exogenous SARS-CoV-2 in wastewater. The ALODs using RT-dPCR were approximately 2-5 times lower than those using RT-qPCR. During sample processing, the endogenous (n = 96) and exogenous (n = 24) SARS-CoV-2 wastewater samples were separated, and RNA was extracted from both wastewater eluates and pellets (solids). The RT-dPCR platform demonstrated a detection rate significantly greater than that of RT-qPCR for the CDC N1 and CDC N2 assays in the eluate (N1, p = 0.0029; N2, p = 0.0003) and pellet (N1, p = 0.0015; N2, p = 0.0067) samples. The positivity results also indicated that for the analysis of SARS-CoV-2 RNA in wastewater, including the eluate and pellet samples may further increase the detection sensitivity using RT-dPCR.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Assay limits of detection for RT-qPCR and RT-dPCR for CDC N1 and N2.
See this image and copyright information in PMC

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