title: Performance assessment PCR-based assays targeting bacteroidales genetic markers of bovine fecal pollution.
abstract: There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from Bacteroidales. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require a priori characterization at each watershed of interest.
authors.affiliation: 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.forename: Orin C
authors.initials: OC
authors.lastname: Shanks
authors.forename: Karen
authors.initials: K
authors.lastname: White
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Kelty
authors.forename: Sam
authors.initials: S
authors.lastname: Hayes
authors.forename: Mano
authors.initials: M
authors.lastname: Sivaganesan
authors.forename: Michael
authors.initials: M
authors.lastname: Jenkins
authors.forename: Manju
authors.initials: M
authors.lastname: Varma
authors.forename: Richard A
authors.initials: RA
authors.lastname: Haugland
mesh_terms: D000818:Animals; D041963:Bacteroidetes / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D002417:Cattle; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D005819:Genetic Markers; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D014873:Water Pollutants
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D005819:Genetic Markers; D012336:RNA, Ribosomal, 16S; D014873:Water Pollutants