Freeze-drying as a novel concentrating method for wastewater detection of SARS-CoV-2

Extracting and detecting viral RNA in wastewater has proven to be a rapid and cost-effective approach for community-level monitoring during the recent global Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic. Various sample concentrating methods, such as centrifugal ultrafiltration, have been utilized in wastewater SARS-CoV-2 detection studies. However, freeze drying, a promising technique commonly used for concentrating and preserving various biological samples, has yet to be explored in this field. This study compared the performance of freeze-drying and the widely used centrifugal ultrafiltration method in terms of recovery rate, detection limit, and other key parameters for concentrating 72 wastewater samples collected from four facilities in El Paso, TX. Statistical analyses revealed that the freeze-drying method demonstrated higher overall recovery efficiency (20.33% vs 13.00%), a superior detection ratio (68.4% vs. 31.6%), and lower detection limits (0.06copies/mL vs 0.35copis/mL) than centrifugal ultrafiltration, particularly during the early stages of the pandemic. Despite its longer processing time than centrifugal ultrafiltration, freeze-drying offers several notable advantages, including eliminating pretreatment steps, providing flexible sample storage options, preventing signal loss and sample degradation, and reducing labor and exposure risks. Freeze-drying also does not require intensive training for concentrating sewage water. These benefits, combined with its efficient capture of viral RNA, position freeze-drying as a promising alternative for wastewater virus detection, especially in resource-constrained lab settings at local wastewater treatment plants. The protocol and findings reported here provide a baseline for further development of freeze-drying-based methods for enabling community-level early warning and surveillance against emerging viral threats in the future.