Influence of storage conditions and multiple freeze-thaw cycles on N1 SARS-CoV-2, PMMoV, and BCoV signal.

Wastewater-based epidemiology/wastewater-based surveillance (WBE/WBS) continues to serve as an effective means of monitoring various diseases, including COVID-19 and the emergence of SARS-CoV-2 variants, at the population level. As the use of WBE expands, storage conditions of wastewater samples will play a critical role in ensuring the accuracy and reproducibility of results. In this study, the impacts of water concentration buffer (WCB), storage temperature, and freeze-thaw cycles on the detection of SARS-CoV-2 and other WBE-related gene targets were examined. Freeze-thawing of concentrated samples did not significantly affect (p > 0.05) crossing/cycle threshold (Ct) value for any of the gene targets studied (SARS-CoV-2 N1, PMMoV, and BCoV). However, use of WCB during concentration resulted in a significant (p < 0.05) decrease in Ct for all targets, and storage at -80 °C (in contrast to -20 °C) appeared preferable for wastewater storage signal stability based on decreased Ct values, although this was only significantly different (p < 0.05) for the BCoV target. Interestingly, when Ct values were converted to gene copies per influent sample, no significant differences (p > 0.05) were observed in any of the targets examined. Stability of RNA targets in concentrated wastewater against freeze-thaw degradation supports archiving of concentrated samples for use in retrospective examination of COVID-19 trends and tracing SARS-CoV-2 variants and potentially other viruses, and provides a starting point for establishing a consistent procedure for specimen collection and storage for the WBE/WBS community.