Optimizing solid-based methods for SARS-CoV-2 detection in wastewater: addressing PCR inhibition and variant challenges.

Wastewater surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is a valuable tool for monitoring coronavirus disease prevalence. However, the dynamics of SARS-CoV-2 and its RNA in wastewater remain largely uncharacterized, leading to uncertainties in viral RNA quantification. This study aimed to improve a recently developed solid-based viral RNA extraction method used in wastewater surveillance programs in Japan. Eighty-four RNA samples extracted from the solid fractions of influent and sludge from two wastewater treatment plants from January to December 2022 were analyzed using reverse transcription quantitative PCR (RT-qPCR) for SARS-CoV-2 and control RNAs. Spiking experiments with a processing control demonstrated that PCR inhibition significantly affected RNA samples from the solid fractions. Contrary to expectations, the commercially available PCR inhibitor removal kit was ineffective, whereas a simple dilution mitigated the inhibitory effect, suggesting the presence of uncharacterized inhibitory factor(s) in the solid fraction. Furthermore, the emergence of the Omicron variant decreased the sensitivity of the CDC N1 RT-qPCR assay. This study demonstrates the improvement of the solid-based method by addressing PCR inhibition and variant emergence and emphasizes the importance of continuous quality control measurements in wastewater surveillance for the effective management of outbreaks caused by evolving viruses, such as SARS-CoV-2.IMPORTANCEThe standardization of methodologies for SARS-CoV-2 wastewater surveillance has not yet been achieved because of the heterogeneous characteristics of wastewater, even within the same country or sewer system. Studies have demonstrated efficient detection of SARS-CoV-2 RNA in the solid fraction of influent wastewater and sludge. A solid-based methodology has recently been developed for the detection of SARS-CoV-2 RNA; however, there remains potential for further refinement. We assessed the effect of RT-qPCR inhibition in samples from the solid fraction of wastewater using virus-like particles (VLPs) as a processing control. This study emphasizes that the methodology for wastewater surveillance should be regularly evaluated with appropriate quality control (QC) measures and refined based on prevailing circumstances.