During the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to detect and quantify SARS-CoV-2 RNA in wastewater, while monitoring viral genome mutations requires separate approaches such as deep genomic sequencing. A high throughput sequencing platform (ATOPlex) that uses a multiplex tiled PCR-based enrichment technique has shown promise in detecting viral variants while also providing virus quantitation data. However, sensitivities of both RT-qPCR and sequencing analyses can be impacted through losses occurring during sample processing (e.g., sample handling, concentration, nucleic acid extraction and RT-qPCR), therefore process limit of detection (PLOD) assessments are needed to estimate the copy numbers of target molecule required to attain specific probability of detection. In this study, we compare the PLOD estimates of four commonly used RT-qPCR assays for quantification of SARS-CoV-2 (US CDC N1 and N2, China CDC N and ORF1ab) to that of ATOPlex sequence analyses through seeding known concentrations of gamma-irradiated SARS-CoV-2 into wastewater. Results suggest that among the RT-qPCR assays, US CDC N1 was the most sensitive, especially at lower SARS-CoV-2 seed levels. However, when results from all RT-qPCR assays were combined, it resulted in the greater detection rates than individual assays, suggesting that application of multiple assays is better suited for detection of trace levels of SARS-CoV-2. Furthermore, while ATOPlex offers promising approach to SARS-CoV-2 wastewater surveillance, this technology is appeared to be less sensitive compared to RT-qPCR and requires further refinements for routine monitoring and quantification of SARS-CoV-2. Nonetheless, the combination of both approaches (RT-qPCR and ATOPlex) may be a powerful tool to simultaneously quantify the viral load in wastewater and monitor emerging variants of concern.
RT-qPCR and ATOPlex sequencing for the sensitive detection of SARS-CoV-2 RNA for wastewater surveillance