1. A novel genetic marker for the rapid detection of Bacteroides fragilis in recreational water as a human-specific faecal indicator.
- Author
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Lee CS, Marion JW, and Lee J
- Subjects
- DNA Primers, Humans, Ohio, Prevotella enzymology, Prevotella genetics, Prevotella isolation & purification, RNA, Ribosomal, 16S genetics, Recreation, Reverse Transcriptase Polymerase Chain Reaction, Sensitivity and Specificity, Bacteroides fragilis enzymology, Bacteroides fragilis genetics, Bacteroides fragilis isolation & purification, DNA Gyrase genetics, Feces microbiology, Fresh Water microbiology, Genetic Markers, Protein Subunits genetics
- Abstract
Bacteroides spp. has gained substantial interest among the suggested potential candidates for alternative faecal indicators for untreated recreational waters by the US EPA. Interest in Bacteroides as a faecal indicator is based upon the relative abundance of selected members of the Bacteroides genus in the human colon and human faeces. In this study, we developed a real-time PCR detection system based on gyrase B subunit genes (gyrB) specific to Bacteroides fragilis. The gryB-based method was compared with previously described 16S rRNA-based real-time qPCR methods and evaluated for specificity, sensitivity and robustness in detecting B. fragilis from untreated recreational water impacted by human and non-human faecal sources. The new gyrB-based system only detected B. fragilis, whereas the 16S rRNA-based methods generated cross-amplifications with other Bacteroides and Prevotella species. We used a procedure of prefiltration, filtration, sonication and DNA concentration in order to improve the DNA extraction efficiency and the sensitivity of the real-time PCR while removing interference. The amplification and sequencing of PCR products generated by the gyrB-based method confirmed that gyrB-amplified sequences only contained B. fragilis. This rapid method is useful for quantifying faecal contamination and may assist beach and watershed managers in elucidating possible contamination sources.
- Published
- 2011
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