Simultaneous microbial capture and nucleic acid extraction from wastewater with minimal pre-processing and high recovery efficiency

Abstract

The COVID-19 pandemic accelerated research towards developing low-cost assays for automated urban wastewater monitoring assay that can be integrated into an environmental surveillance system for early warning of frequent disease outbreaks and future pandemics. Microbial concentration is one of the most challenging steps in wastewater surveillance, due to the sample heterogeneity and low pathogen load. Keeping in mind the requirements of large-scale testing in densely populated low- or middle-income countries (LMICs), such assays would need to be low-cost and have rapid turnaround time with high recovery efficiency. In this study, two such methods are presented and evaluated against commercially available kits for pathogen detection in wastewater. The first method utilizes paper dipsticks while the second method comprises of a PTFE membrane filter (PMF) integrated with a peristaltic pump. Both methods were used to concentrate and isolate nucleic acids from different microbes such as SARS-CoV-2, pepper mild mottle virus (PMMoV), bacteriophage Phi6, and E. coli from wastewater samples with minimal or no sample pre-processing. While the paper dipstick method is suitable for sub-milliliter sample volume, the PMF method can be used with larger volumes of wastewater sample (40 mL) and can detect multiple microbes with recovery efficiency comparable to commercially available kits.