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

Jean E T McLain, Hodon Ryu, Leila Kabiri-Badr, Channah M. Rock, Morteza Abbaszadegan

Research output: Contribution to journalArticle

18 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)38-43
Number of pages6
JournalFEMS Microbiology Letters
Volume299
Issue number1
DOIs
StatePublished - Oct 2009

Fingerprint

Bacteroides
Gene Amplification
rRNA Genes
Feces
Fishes
Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
DNA
DNA Primers
Fresh Water
Clone Cells
Bacteria
Water

Keywords

  • Bacteroides
  • Microbial source tracking
  • Water quality

ASJC Scopus subject areas

  • Microbiology
  • Genetics
  • Molecular Biology

Cite this

Lack of specificity for PCR assays targeting human Bacteroides 16S rRNA gene : Cross-amplification with fish feces. / McLain, Jean E T; Ryu, Hodon; Kabiri-Badr, Leila; Rock, Channah M.; Abbaszadegan, Morteza.

In: FEMS Microbiology Letters, Vol. 299, No. 1, 10.2009, p. 38-43.

Research output: Contribution to journalArticle

McLain, Jean E T ; Ryu, Hodon ; Kabiri-Badr, Leila ; Rock, Channah M. ; Abbaszadegan, Morteza. / Lack of specificity for PCR assays targeting human Bacteroides 16S rRNA gene : Cross-amplification with fish feces. In: FEMS Microbiology Letters. 2009 ; Vol. 299, No. 1. pp. 38-43.
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