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

Warish Ahmed; Wendy J M Smith; Suzanne Metcalfe; Greg Jackson; Phil M Choi; Mary Morrison; Daniel Field; Pradip Gyawali; Aaron Bivins; Kyle Bibby; Stuart L Simpson

doi:10.1021/acsestwater.1c00387

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

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

Authors

Affiliations

¹ CSIRO Land and Water, Ecosciences Precinct, 41 Boggo Road, Dutton Park, QLD 4102, Australia.
² Water Unit, Health Protection Branch, Prevention Division, Queensland Health, Brisbane, QLD 4001, Australia.
³ Institute of Environmental Science and Research Ltd. (ESR), Porirua 5240, New Zealand.
⁴ Department of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame, Notre Dame, Indiana 46556, United States.
⁵ CSIRO Land and Water, Lucas Heights, NSW 2234, Australia.

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.