Freeze-drying as a Novel Concentrating Method for Wastewater Detection of SARS-CoV-2

Detecting viral RNA from wastewater has emerged as a cost-effective approach for community-level surveillance during the recent SARS-CoV-2 pandemic. Although various concentrating methods have been developed, none are optimal for all key requirements for wastewater viral detection. Freeze-drying, a technique widely used for concentrating and preserving biological materials, remains underexplored for this purpose. This study compared the performance of freeze-drying and centrifugal ultrafiltration in terms of recovery efficiency, detection limit, and other key parameters. Early pandemic samples in this study, with extremely low viral concentrations, offered an ideal benchmark to assess their suitability for early-warning applications. Statistical analyses showed that freeze-drying achieved significantly higher recovery efficiency (20.2 ± 3.8% vs. 12.9 ± 2.0%), superior detection ratio (68.4% vs. 31.6%), and lower detection limit (0.06 vs. 0.35 copies/mL) compared to centrifugal ultrafiltration. To our knowledge, this is the first study to apply freeze-drying for wastewater-based viral detection. Despite its longer processing time, freeze-drying offers multiple advantages, including elimination of pretreatment steps, flexible storage, reduced RNA degradation under cryogenic conditions, minimal pathogen exposure, lower labor demands, and less human interference during processing. These features position freeze-drying as a novel alternative for wastewater-based viral surveillance, particularly for developing early-warning systems in resource-limited settings.