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ARS Home » Pacific West Area » Maricopa, Arizona » U.S. Arid Land Agricultural Research Center » Water Management and Conservation Research » Research » Publications at this Location » Publication #232310
Title: Lack of specificity for PCR assays targeting human Bacteroides 16S rRNA gene: cross-amplification with fish feces

Author
item McLain, Jean
item RYU, HODON - NSF, ASU, TEMPE, AZ
item KABIRI-BADR, LEILA - NSF, ASU, TEMPE, AZ
item ROCK, CHANNAH - UNIV OF AZ, TUCSON
item ABBASZADEGAN, MORTEZA - NSF, ASU, TEMPE, AZ

Submitted to: FEMS Microbiology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/3/2009
Publication Date: 8/14/2009
Citation: Mclain, J.E., Ryu, H., Kabiri-Badr, L., Rock, C.M., Abbaszadegan, M. 2009. Lack of specificity for PCR assays targeting human Bacteroides 16S rRNA gene: cross-amplification with fish feces. FEMS Microbiology Letters. p. 38-43.

Interpretive Summary: The increasing trend worldwide to reuse of water resources, both in urban and rural environments, elevates the importance of distinguishing the sources of fecal contamination in surface waters. Microbial source tracking methods focused on members of the genus Bacteroides have been increasingly utilized in identifying and quantifying fecal contamination sources. We found significant cross-amplification of a widely used human-specific Bacteroides 16S rRNA molecular marker with fecal DNA from two fish species. Further work confirmed that, although the fish fecal bacteria contained the human markers, the fish organisms were not the same as human, but were unique strains, presumptively of the genus Bacteroides. Our results strongly demonstrated the potential for cross-amplification of human-specific markers with fish feces, and may call into question the use of published Bacteroides-specific molecular markers in quantification of human fecal contamination in waters where fish contribute to fecal inputs.

Technical Abstract: Methods focused on members of the genus Bacteroides have been increasingly utilized in microbial source tracking studies for identifying and quantifying sources of non-point fecal contamination. We present results using real-time PCR to show significant cross-amplification of a human-specific Bacteroides 16S rRNA molecular marker with fecal DNA from two fish species. Sequencing of PCR amplicons generated from fish fecal DNA revealed no mismatches to the human-specific primers and probe, but the nucleotide sequences of clones from fish fecal samples differed markedly from those of human feces, suggesting that the fish-related bacteria are unique strains, presumptively of the genus Bacteroides. Our results strongly demonstrate the potential for cross-amplification of human-specific markers with fish feces, and may call into question the use of published Bacteroides-specific molecular markers in quantification of human fecal contamination in waters where fish may contribute to fecal inputs.