title: Evaluation of genetic markers from the 16S rRNA gene V2 region for use in quantitative detection of selected Bacteroidales species and human fecal waste by qPCR.
abstract: Molecular methods for quantifying defined Bacteroidales species from the human gastrointestinal tract may have important clinical and environmental applications, ranging from diagnosis of infections to fecal source tracking in surface waters. In this study, sequences from the V2 region of the small subunit ribosomal RNA gene were targeted in the development of qPCR assays to quantify DNA from six Bacteroides and one Prevotella species. In silico and experimental analyses suggested that each of the assays was highly discriminatory in detecting DNA from the intended species. Analytical sensitivity, precision and ranges of quantification were demonstrated for each assay by coefficients of variation of less than 2% for cycle threshold measurements over a range from 10 to 4×10(4) target sequence copies. The assays were applied to assess the occurrence and relative abundance of their target sequences in feces from humans and five animal groups as well as in 14 sewage samples from 13 different treatment facilities. Sequences from each of the species were detected at high levels (>10(3)copies/ng total extracted DNA) in human wastes. Sequences were also detected by each assay in all sewage samples and, with exception of the Prevotella sequences, showed highly correlated (R(2)≥0.7) variations in concentrations between samples. In contrast, the occurrence and relative abundance profiles of these sequences differed substantially in the fecal samples from each of the animal groups. These results suggest that analyses for multiple individual Bacteroidales species may be useful in identifying human fecal pollution in environmental waters.
authors.affiliation: US Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, Cincinnati, OH 45268, USA. haugland.rich@epa.gov
authors.forename: Richard A
authors.initials: RA
authors.lastname: Haugland
authors.forename: Manju
authors.initials: M
authors.lastname: Varma
authors.forename: Mano
authors.initials: M
authors.lastname: Sivaganesan
authors.forename: Catherine
authors.initials: C
authors.lastname: Kelty
authors.forename: Lindsay
authors.initials: L
authors.lastname: Peed
authors.forename: Orin C
authors.initials: OC
authors.lastname: Shanks
mesh_terms: D000818:Animals; D058491:Bacterial Load / Q000379:methods; D041963:Bacteroidetes / Q000235:genetics / Q000302:isolation & purification*; D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D006801:Humans; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D018720:Prevotella / Q000235:genetics / Q000302:isolation & purification*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D012722:Sewage / Q000382:microbiology
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012722:Sewage