Gene

Wastewater

Real-time polymerase chain reaction

Digital polymerase chain reaction

Molecular biology

Biology

Messenger RNA

Chromatography

Virology

Biochemistry

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

Reverse transcription polymerase chain reaction

Coronavirus disease 2019 (COVID-19)

Environmental engineering

Infectious disease (medical specialty)

Environmental science

Medicine

Chemistry

Polymerase chain reaction

Detection limit

Disease

Pathology

ACS ES&T Water

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.

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