Quantitative and Viability-Based Detection of Pathogenic Bacteria in Environmental Waters by Full-Length 16S rRNA Sequencing
The project aimed to simultaneously detect and quantify pathogenic bacterial species in environmental waters based on full-length 16S rRNA gene sequencing. To achieve this, a quantitative sequencing (qSeq) method using internal standard DNA spiking was developed. In addition, cis-dichlorodiammine platinum (CDDP), a membrane integrity-based viability dye, was evaluated for the selective detection of viable bacteria. Specific experiments were conducted to: (1) Compare the responses of bacterial communities in environmental waters to two viability dyes, PMAxx and cis-dichlorodiammine platinum (CDDP). (2) Optimize qSeq analytical conditions using the NBRC Microbial DNA Cocktail and ZymoBIOMICS Microbial Community DNA Standard II (Log Distribution). (3) Apply CDDP-qSeq and qSeq to river waters, including those receiving minimally treated WWTP discharges, for the simultaneous detection and quantification of multiple viable pathogenic bacterial species.