title: Quantitative real-time PCR detection of oral Enterococcus faecalis in humans.
abstract: OBJECTIVE
  Enterococcus faecalis is consistently associated with recurrent root canal infections. Only low concentrations of E. faecalis in the human mouth have been demonstrated by culture techniques. Quantitative detection strategies more sensitive than culturing, such as quantitative PCR (qPCR), could provide more illuminating data.
  
  
  METHODS
  Thirty outpatients attending the University of Michigan Graduate Endodontic Clinic for endodontic treatment provided oral rinse samples that were analysed for E. faecalis using qPCR and microbiological culturing. A SYBR Green I qPCR protocol was developed for the quantifiable detection of E. faecalis and total bacteria in oral rinse samples using primers designed to target the 16S rRNA gene. Annealing temperature and primer, magnesium ion, and dimethyl sulfoxide concentrations were investigated for optimisation of the protocol; a minimum sensitivity limit of 23 rRNA copies (an estimated six E. faecalis cells) was established for E. faecalis in pure culture, and 104 rRNA copies (an estimated 26 E. faecalis cells) in mixed culture.
  
  
  RESULTS
  In qPCR assays, based on extrapolation from estimated rRNA gene copy numbers, E. faecalis comprised 0.0006-0.0047% of a total bacteria load that ranged from 5.92 x 10(5) to 5.69 x 10(7) cells/ml of oral rinse. E. faecalis was detected in five (17%) samples in concentrations from 114 to 490 cells/ml. In parallel culture assays E. faecalis were detected in only two samples (7%) of the five identified by qPCR and in concentrations 30 and 240 CFU/ml.
  
  
  CONCLUSIONS
  qPCR reported a higher incidence of E. faecalis in oral rinse samples than culture techniques and afforded greater sensitivity.
authors.affiliation: Department of Cariology, Restorative Sciences, and Endodontics, University of Michigan, Ann Arbor, MI 48109-1078, USA. csedgley@umich.edu
authors.forename: C M
authors.initials: CM
authors.lastname: Sedgley
authors.forename: A C
authors.initials: AC
authors.lastname: Nagel
authors.forename: C E
authors.initials: CE
authors.lastname: Shelburne
authors.forename: D B
authors.initials: DB
authors.lastname: Clewell
authors.forename: O
authors.initials: O
authors.lastname: Appelbe
authors.forename: A
authors.initials: A
authors.lastname: Molander
mesh_terms: D003114:Colony-Forming Units Assay; D004269:DNA, Bacterial / Q000032:analysis; D013293:Enterococcus faecalis / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009055:Mouth / Q000382:microbiology*; D015347:RNA Probes; D012329:RNA, Bacterial / Q000032:analysis*; D012336:RNA, Ribosomal, 16S; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial; D015347:RNA Probes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S