Simultaneous extraction and detection of DNA and RNA from viruses, prokaryotes, and eukaryotes in wastewater using a...

Wastewater surveillance can offer a comprehensive grasp of infectious disease prevalence and human health because wastewater contains various human-derived microbial pathogens, including viruses, bacteria, and fungi. However, methods capable of simultaneous detection of multiple groups of targets in the automated systems and large-scale surveillance are still under development. Here, we demonstrated the modification, involving the addition of bead-beating, to the existing COPMAN (COagulation and Proteolysis method using MAgnetic beads for detection of Nucleic acids in wastewater) enabled enhanced detection of various microorganisms, including SARS-CoV-2. The modified method, termed bead-beating COPMAN (BB-COPMAN), was evaluated through spike-and-recovery experiments and comparative analysis against three previously reported methods for simultaneous DNA/RNA detection. Our study targeted a range of microorganisms, including enveloped and non-enveloped RNA viruses (SARS-CoV-2, PMMoV), a DNA virus (crAssphage), archaea, gram-negative and gram-positive bacteria (E. coli, Lachnospiraceae), antibiotic resistance gene (ampC), and fungi (Candida albicans). The recovery rates of BB-COPMAN for gram-negative and gram-positive bacteria were 17 and 2.1-fold higher, respectively, compared to the method for DNA/RNA detection. Additionally, BB-COPMAN exhibited the highest extraction efficiency among the tested methods, achieving 1.2-5.7 times more DNA and 1.1-69 times more RNA yield on average. BB-COPMAN allowed the detection of SARS-CoV-2 from all nine samples and PMMoV at concentrations 39-97 times higher than other methods. Moreover, BB-COPMAN detected larger amounts of DNA for four out of six DNA targets than the previously reported DNA/RNA detection method. These results demonstrated that BB-COPMAN enables enhanced detection of multiple targets in a single flow of nucleic acid extraction, making the method well-suited for automated systems. In conclusion, BB-COPMAN is a promising method in wastewater surveillance for assessing the prevalence of wide range of pathogenic microorganisms.