title: A novel genetic marker for the rapid detection of Bacteroides fragilis in recreational water as a human-specific faecal indicator.
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.
authors.affiliation: College of Public Health, Division of Environmental Health Sciences, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Chang Soo
authors.initials: CS
authors.lastname: Lee
authors.forename: Jason W
authors.initials: JW
authors.lastname: Marion
authors.forename: Jiyoung
authors.initials: J
authors.lastname: Lee
mesh_terms: D001441:Bacteroides fragilis / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification*; D027081:DNA Gyrase / Q000235:genetics*; D017931:DNA Primers; D005243:Feces / Q000382:microbiology*; D005618:Fresh Water / Q000382:microbiology*; D005819:Genetic Markers; D006801:Humans; D009820:Ohio; D018720:Prevotella / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D021122:Protein Subunits / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011998:Recreation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D005819:Genetic Markers; D021122:Protein Subunits; D012336:RNA, Ribosomal, 16S; D027081:DNA Gyrase