posted on 2024-03-01, 20:12authored bySarah Schurig, Arianna Ceruti, Andy Wende, Phillip Lübcke, Elias Eger, Katharina Schaufler, Michael Frimpong, Uwe Truyen, Rea Maja Kobialka, Ahmed Abd El Wahed
Wastewater systems
represent a threat for the transmission of pathogens
in the case of improper treatment. Systematic surveillance based on
wastewater is important as an early warning system, e.g., COVID-19.
However, current procedures for microorganism identification are often
not applicable at the point-of-need. In this study, a rapid and portable
DNA extraction protocol was developed and verified to identify the
microbial wastewater composition by nanopore sequencing. The study
compared different pretreatment and buffer options combined with the
reverse purification principle. Extraction efficacy was assessed using Staphylococcus aureus and Escherichia
coli spiked wastewater and crude samples from different
collection points at the treatment plant. Results were compared to
a spin column-based reference method and a real-time polymerase chain
reaction. Lysis buffer combined with bead beating showed the best
extraction efficacies using an Oxford nanopore. It demonstrated an
adequate sensitivity of down to 103 copies. Up to eight
different bacterial species could be identified at each collection
point by nanopore sequencing. The developed protocol achieved a 3-fold
increase of average length, an increase of quality score, and a 15-fold
decrease of total reads. The evaluated molecular detection methods
are a useful tool for the analysis of bacteria within less than 3.5
h and could contribute to public health monitoring of wastewater.