title: Performance of PCR-based assays targeting Bacteroidales genetic markers of human fecal pollution in sewage and fecal samples.
abstract: There are numerous PCR-based assays available to characterize human fecal pollution in ambient waters. Each assay employs distinct oligonucleotides and many target different genes and microorganisms leading to potential variations in assay performance. Performance comparisons utilizing feces and raw sewage samples are needed to determine which assays are best suited for expensive and time-consuming field validation, fate, transport, and epidemiology studies. We report the assessment of five end-point PCR and 10 real-time quantitative PCR (qPCR) assays that target genes from presumptive Bacteroidales microorganisms reported to be associated with human feces. Each assay was tested against a reference collection of 54 primary influent sewage samples collected from different geographical locations across the United States and 174 fecal DNA extracts from 23 different animal sources. Experiments indicate that human-associated genetic markers are distributed across a broad range of human populations but show substantial differences in specificity for human feces suggesting that particular assays may be more suitable than others depending on the abundance of genetic marker required for detection and the animal sources impacting a particular watershed or beach of interest.
authors: U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, 26 West Martin Luther King Drive, Cincinnati, Ohio 45268, USA. shanks.orin@epa.gov
authors: Orin C
authors: OC
authors: Shanks
authors: Karen
authors: K
authors: White
authors: Catherine A
authors: CA
authors: Kelty
authors: Mano
authors: M
authors: Sivaganesan
authors: Janet
authors: J
authors: Blannon
authors: Mark
authors: M
authors: Meckes
authors: Manju
authors: M
authors: Varma
authors: Richard A
authors: RA
authors: Haugland
mesh_terms: D041963:Bacteroidetes / Q000235:genetics*; D001681:Biological Assay / Q000379:methods* / Q000592:standards; D002138:Calibration; D004787:Environmental Pollution / Q000032:analysis*; D005243:Feces / Q000382:microbiology*; D005819:Genetic Markers; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D011786:Quality Control; D012722:Sewage / Q000382:microbiology*
chemical_list: D005819:Genetic Markers; D012722:Sewage