Maximizing viral nucleic acid yield from passive samplers: Evaluating elution and extraction protocols

The COVID-19 pandemic has underscored the need for effective viral tracking in aqueous environments, particularly for non-enteric viruses. Despite advances in wastewater monitoring, surveillance of viruses in freshwater remains limited due to traditional sampling challenges. This study refines GAC-based passive sampling protocols by determining optimal extraction and elution methods for enhancing the recovery of viral nucleic acids in freshwater. Three commercially available total nucleic acid (TNA) extraction kits and four elution buffers were assessed for their ability to recover SARS-CoV-2 and bacteriophage MS2 from GAC-based samplers. The Promega Wizard® Enviro Total Nucleic Acid Kit, paired with a Tween®20-based buffer, provided the highest virus recovery efficiency for GAC-based passive sampling. Field-scale applications demonstrated the effectiveness of GAC-based passive samplers in capturing SARS-CoV-2, INFA, RSV, and MeV using the optimized protocols. The combination of the Tween®20 based buffer and the Promega kit led to increased detection frequencies in grab samples, which remained lower in recovery than passive sampling. This study underscores the importance of selecting appropriate TNA extraction kits and elution buffers to maximize virus recovery from passive samples. By optimizing these protocols, we enhance the sensitivity and reliability of viral surveillance in freshwater ecosystems.