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

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.

Monitoring SARS-CoV-2 RNA in Wastewater with RT-qPCR and Chip-Based RT-dPCR: Sewershed-Level Trends and Relationships to COVID-19

“…PCR platform could impact measured SARS-CoV-2 concentrations due to differences between quantification methods and limits of detection. 8 , 21 Calculated ALOD values for N1 and N2 uniplex and duplex RT-qPCR assays were 21.8 and 24.8 copies/reaction, which are around 6–7 times greater than the probabilistic ALOD determined by the Poisson distribution (3 copies/reaction) and ∼20 times greater than the theoretical ALOD (1 copy/reaction). 15 Our ALODs for uniplex and duplex N2 are comparable to the uniplex RT-qPCR N2 ALOD (26.7 copies/reaction) but our N1 ALOD is higher than the N1 ALOD value (9.5 copies/reaction) previously reported.…”

“…Since wastewater surveillance was used for monitoring COVID-19 from the start of the pandemic, 4 , 5 methodologies have varied greatly. 6 − 8 Numerous research groups have demonstrated that SARS-CoV-2 in wastewater reflects trends in community-level COVID-19 dynamics, and some reports observe that wastewater trends precede those of clinical reports. 1 , 9 Molecular-based wastewater surveillance depends on many factors, such as molecular processing methods (e.g., concentration and extraction of viral RNA), biochemical and microbiological complexity of wastewater matrix that can cause inhibition of molecular analyses, residence time in the sewer system, composite sampling duration and frequency, upstream sample processing methods, and virus recovery.…”

“… 24 Accurate quantification can be achieved by optimizing primer and probe sets for each target, but increasing number of targets can decrease multiplex assay performance (e.g., efficiency and limit of detection). 17 , 23 Previously, researchers have employed both digital droplet PCR 13 , 25 and chip-based dPCR 8 for wastewater surveillance of SARS-CoV-2 RNA. However, few studies report the use of chip-based dPCR for wastewater monitoring.…”

“… 1 , 24 Recently, RT-qPCR and RT-dPCR platforms were compared for trace detection of SARS-CoV-2 RNA in wastewater. 8 However, multiplex, chip-based RT-dPCR has not yet been evaluated for wastewater surveillance of SARS-CoV-2 RNA.…”

Advances in virus detection methods for wastewater-based epidemiological applications

“…The main advantages of the RT-dPCR over the RT-qPCR are its non-reliance on the calibration curve for direct absolute quantification, and reduced effects of PCR inhibitors [ 29 ]. Greater analytical sensitivity was observed in the RT-dPCR for the detection of SARS-COV-2 in clinical samples [ 30 ] and in wastewater samples [ 9 , 18 , 19 ]. In the study by Ref.…”