Lack of specificity for PCR assays targeting human Bacteroides 16S rRNA gene: cross-amplification with fish feces

Authors


  • Editor: Jan-Ulrich Kreft

Correspondence: Jean E.T. McLain, USDA-ARS, US Arid Land Agricultural Research Center, 21881 N Cardon Lane, Maricopa, AZ 85138, USA. Tel.: +1 520 316 6378; fax: +1 520 316 6330; e-mail: jean.mclain@ars.usda.gov

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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