title: Lack of specificity for PCR assays targeting human Bacteroides 16S rRNA gene: cross-amplification with fish feces.
abstract: Methods focused on members of the genus Bacteroides have been increasingly utilized in microbial source-tracking studies for identifying and quantifying sources of nonpoint fecal contamination. We present results using standard and real-time PCR to show cross-amplification of Bacteroides 16S rRNA gene molecular assays targeting human fecal pollution with fecal DNA from freshwater fish species. All except one of the presumptively human-specific assays amplified fecal DNA from at least one fish species, and one real-time PCR assay amplified DNA from all fish species tested. Sequencing of PCR amplicons generated from fish fecal DNA using primers from the real-time assay revealed no mismatches to the human-specific probe sequences, but the nucleotide sequences of clones from fish fecal samples differed markedly from those of human feces, suggesting that the fish-related bacteria may be different strains. Our results strongly demonstrate the potential for cross-amplification of human-specific PCR assays with fish feces, and may call into question the results of studies in which these Bacteroides-specific molecular markers are used to quantify human fecal contamination in waters where fish contribute to fecal inputs.
authors.affiliation: USDA-ARS, US Arid Land Agricultural Research Center, Maricopa, AZ 85138, USA. jean.mclain@ars.usda.gov
authors.forename: Jean E T
authors.initials: JE
authors.lastname: McLain
authors.forename: Hodon
authors.initials: H
authors.lastname: Ryu
authors.forename: Leila
authors.initials: L
authors.lastname: Kabiri-Badr
authors.forename: Channah M
authors.initials: CM
authors.lastname: Rock
authors.forename: Morteza
authors.initials: M
authors.lastname: Abbaszadegan
mesh_terms: D000818:Animals; D001439:Bacteroides / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000235:genetics*; D005243:Feces / Q000382:microbiology*; D005399:Fishes / Q000382:microbiology; D005618:Fresh Water / Q000382:microbiology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D013045:Species Specificity
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S