Microbubbles for improved nucleic acid extraction in wastewater samples for viral RNA detection

The use of wastewater-based epidemiology has increased in recent years due to the publication of COVID-19 online trackers and the focus of the media on the pandemic. Yet the analysis of viromes in wastewater has been widely applied for several decades in conjunction with traditional chemical analysis approaches. However, even though real time quantitative polymerase chain reaction (RT-qPCR) based molecular detection methods are now mainstream in large and small labs alike, wastewater sampling and nucleic acid extraction procedures are not yet standardized or optimized to enable routine and robust analysis and results interpretation. Here, we employ a flotation-based nucleic acid extraction method using microbubbles that allows for simple direct collection and lysis of total wastewater samples without the requirement for pasteurization or filtration of solid components prior to analysis. An additional advantage discovered during testing was reduced sample input needs while maintaining sensitivity compared to precipitation and ultrafiltration-based methods. Microbubbles designed to bind nucleic acids enable convenient workflows, fast extraction, and concentration and purification of RNA and DNA that is compatible with downstream genomic analyses. SUMMARY Microbubble-based capture of nucleic acids from raw (unpasteurized) and unfiltered (containing solids) wastewater with subsequent elution offers several advantages over existing methods. Using microbubbles, the required sample input and protocol duration are reduced while sensitivity of downstream genomic analysis is increased. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study did not receive any funding ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present work are contained in the manuscript