JMIR Publications

ABSTRACT

Background:

Many countries have applied wastewater surveillance of the COVID-19 pandemic to their national public health monitoring measures. The most used methods for the detection of SARS-CoV-2 in wastewater are RT-qPCR and RT-ddPCR. Previous studies have produced conflicting results, thus more research on the subject is required.

Objective:

This research was conducted to further study RT-qPCR and RT-ddPCR for the detection of SARS-CoV-2 in wastewater. In addition, the aim was to study the effect that changes in the analysis pipeline can have on the results. Furthermore, one of the objectives was to find a detection method for low-resource settings.

Methods:

We compared two RT-qPCR kits and RT-ddPCR based on sensitivity, variability, and correlation of SARS-CoV-2 gene copy numbers in wastewater to the incidence of COVID-19. RNA was extracted from the samples with column- and magnetic bead-based RNA extraction methods. SARS-CoV-2 was detected from the samples using N1 and N2 target gene assays for RT-qPCR and N1 and E target gene assays for RT-ddPCR. RT-SIBA was used to detect SARS-CoV-2 from wastewater qualitatively.

Results:

Our results indicate that the most sensitive method to detect SARS-CoV-2 in wastewater was RT-ddPCR. It had the highest positivity rate (26/30), and its limit of detection was the lowest (0.06 gene copies/µl). However, we obtained the best correlation between COVID-19 incidence and SARS-CoV-2 gene copy number in wastewater using RT-qPCR (CC = 0.697, P<.001). Changes in the analysis pipeline were shown to effect the results. We found a significant difference in sensitivity between the two RT-qPCR kits, one having a significantly lower limit of detection and a higher positivity rate than the other. Furthermore, the N1 target gene assay was the most sensitive for both RT-qPCR kits, while no significant difference was found between the tested targets using RT-ddPCR. In addition, the use of different RNA extraction kits affected the result when using the more sensitive RT-qPCR kit. RT-SIBA was able to detect SARS-CoV-2 RNA in wastewater.

Conclusions:

Currently, wastewater surveillance of SARS-CoV-2 is mostly performed with RT-qPCR. Yet, this study, as most of the previous studies, showed RT-ddPCR to be more sensitive. The use of RT-ddPCR in the wastewater surveillance of SARS-CoV-2 should be considered, especially if the amount of SARS-CoV-2 circulating in the population is low. All the analysis steps must be optimized for wastewater surveillance as our study showed that the compatibility of the RNA extraction, the RT-PCR kit and the target gene assay influences the results. In addition, our study showed that RT-SIBA could be used to detect SARS-CoV-2 in wastewater if a qualitative result is sufficient.