title: Evaluation of the Propidium Monoazide-quantitative Polymerase Chain Reaction Method for the Detection of Viable Enterococcus faecalis.
abstract: BACKGROUND
  One limitation of DNA-based molecular assays is their inability to distinguish between live and dead cells. A sample treatment with propidium monoazide (PMA) before DNA amplification has been proposed to overcome this problem. The aim of this in vitro study was to test different concentrations of PMA coupled with quantitative polymerase chain reaction (qPCR) for the detection of viable Enterococcus faecalis.
  
  
  METHODS
  Viable or heat-killed suspensions of E. faecalis (10⁶ colony-forming units/mL) were treated with PMA at 10, 50, and 100 μg/mL before DNA extraction. qPCR was performed using primers complementary for E. faecalis 16S ribosomal RNA sequence. PMA was also tested on bacteria suspensions containing different proportions of viable and dead cells. Bacterial suspensions without PMA treatment were used as positive controls.
  
  
  RESULTS
  The treatment of heat-killed suspensions with PMA at different concentrations significantly reduced the DNA amplification when compared with the group without treatment (P < .0001), indicating that DNA from dead cells was not used as templates. The greatest reduction in qPCR amplification of dead cell DNA was found when 100 μg/mL PMA was used (P < .005). In mixtures containing live/dead cells, PMA allowed selective detection of viable cells.
  
  
  CONCLUSIONS
  PMA was effective in inhibiting qPCR amplification from the DNA of dead cells, enabling in vitro detection and quantification of viable cells of E. faecalis.
authors.affiliation: Discipline of Endodontics, Department of Dentistry, School of Dentistry, University of São Paulo, São Paulo, Brazil. Electronic address: erickapinheiro@usp.br.
authors.forename: Ericka T
authors.initials: ET
authors.lastname: Pinheiro
authors.affiliation: Discipline of Endodontics, Department of Dentistry, School of Dentistry, University of São Paulo, São Paulo, Brazil.
authors.forename: Viviane D
authors.initials: VD
authors.lastname: Neves
authors.affiliation: Discipline of Endodontics, Department of Dentistry, School of Dentistry, University of São Paulo, São Paulo, Brazil.
authors.forename: Caroline C
authors.initials: CC
authors.lastname: Reis
authors.affiliation: Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: Priscila L
authors.initials: PL
authors.lastname: Longo
authors.affiliation: Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: Márcia P A
authors.initials: MP
authors.lastname: Mayer
mesh_terms: D001386:Azides / Q000494:pharmacology*; D058491:Bacterial Load; D001431:Bacteriological Techniques; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D013293:Enterococcus faecalis / Q000302:isolation & purification*; D066298:In Vitro Techniques; D011419:Propidium / Q000031:analogs & derivatives* / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D001386:Azides; D017931:DNA Primers; D004269:DNA, Bacterial; C533957:propidium monoazide; D011419:Propidium
keywords: Endodontic; Enterococcus faecalis; propidium monoazide; quantitative polymerase chain reaction