Open AccessLack of specificity for PCR assays targeting human Bacteroides 16S rRNA gene: cross‐amplification with fish feces
Author(s) -
McLain Jean E.T.,
Ryu Hodon,
KabiriBadr Leila,
Rock Channah M.,
Abbaszadegan Morteza
Publication year - 2009
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2009.01745.x
Subject(s) - biology , feces , bacteroides , amplicon , human feces , fecal coliform , microbiology and biotechnology , 16s ribosomal rna , bacteroidaceae , ribosomal rna , polymerase chain reaction , dna extraction , bacteria , gene , genetics , ecology , water quality
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.