title: Development and application of a polymerase chain reaction assay for the detection and enumeration of bile acid 7alpha-dehydroxylating bacteria in human feces.
abstract: BACKGROUND
  Secondary bile acids are synthesized in the human colon from the bacterial 7alpha-dehydroxylation of primary bile acids. Increased levels of secondary bile acids have been correlated with an increased risk of colon cancer and cholesterol gallstone disease. Based on 16s rDNA sequence analysis, stock cultures of bacterial strains with bile acid 7alpha-dehydroxylation all belong to the genus Clostridium.
  
  
  METHODS
  We developed a polymerase chain reaction (PCR)-based assay that can specifically amplify the baiCD gene, specific to bile acid 7alpha-dehydroxylation pathway. The PCR primer pair was designed using known nucleotide sequences from two different baiCD genes described for Clostridium scindens VPI 12708 and Clostridium hiranonis TO931. Although the DNA sequences of these genes were <70% identical, several regions were conserved enough to design primers with little or no redundancy.
  
  
  RESULTS
  The PCR assay was effective in detecting the baiCD gene in several strains known to exhibit bile acid 7alpha-dehydroxylation activity. The PCR assay also detected the baiCD gene in DNA extracted from fecal dilution series and correlated with the levels of cholic acid 7alpha-dehydroxylating bacteria detected by activity assays. The PCR assay was sensitive enough to detect the baiCD gene in DNA samples extracted directly from as little as 0.5 mg feces.
  
  
  CONCLUSIONS
  This new method should be useful for the monitoring of levels of bile acid 7alpha-dehydroxylating bacteria in human fecal samples.
authors.affiliation: Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, PO Box 980678, Richmond, VA 23298-0678, USA.
authors.forename: James E
authors.initials: JE
authors.lastname: Wells
authors.forename: Kristopher B
authors.initials: KB
authors.lastname: Williams
authors.forename: Terence R
authors.initials: TR
authors.lastname: Whitehead
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Heuman
authors.forename: Phillip B
authors.initials: PB
authors.lastname: Hylemon
mesh_terms: D002793:Cholic Acids / Q000378:metabolism*; D003013:Clostridium / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D006801:Humans; D006900:Hydroxylation; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
chemical_list: D002793:Cholic Acids; D017931:DNA Primers; D012336:RNA, Ribosomal, 16S