Lack of specificity for PCR assays targeting human Bacteroides 16S rRNA gene: cross-amplification with fish feces |
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Authors: | Jean ET McLain Hodon Ryu Leila Kabiri-Badr Channah M Rock & Morteza Abbaszadegan |
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Institution: | USDA-ARS, US Arid Land Agricultural Research Center, Maricopa, AZ, USA;;National Risk Management Research Laboratory, US Environmental Protection Agency, Cincinnati, OH, USA;;National Science Foundation Water Quality Center, Ira A. Fulton School of Engineering, Arizona State University, Tempe, AZ, USA;and;University of Arizona Maricopa Agricultural Center, Maricopa, AZ, USA |
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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. |
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Keywords: | microbial source tracking Bacteroides water quality |
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