title: RNA-based Assay Demonstrated Enterococcus faecalis Metabolic Activity after Chemomechanical Procedures.
abstract: BACKGROUND
  Because ribosomal RNA (rRNA) indicates metabolic cell activity, this study aimed to evaluate the sensitivity of rRNA-based quantitative polymerase chain reaction (RT-qPCR) for the identification of active Enterococcus faecalis in root canals samples compared with a method based on ribosomal DNA (rDNA) (rRNA genes).
  
  
  METHODS
  Samples were taken from 18 teeth with persistent/secondary intraradicular infection before (S1) and after (S2) chemomechanical preparation. RNA and DNA were extracted, and complementary DNA was synthesized from RNA using RT-PCR. Complementary DNA and genomic DNA were subjected to quantitative polymerase chain reaction with primers complementary for E. faecalis 16S rRNA sequence.
  
  
  RESULTS
  E. faecalis was detected in 77.8% and 72.2% of S1 samples using rRNA- and rDNA-based assays, respectively. In contrast, E. faecalis was detected in only 33.3% of S2 samples using rDNA as the template compared with 61.1% using the rRNA-based method. The median concentration of rRNA copies of E. faecalis was significantly higher than rDNA copies, indicating a higher sensitivity for the method targeting rRNA in both S1 (P < .01) and S2 samples (P < .05). After chemomechanical preparation, the number of rRNA and rDNA copies was significantly reduced (P < .05). The high ratio of rRNA to rDNA copies in S2 samples suggested that active E. faecalis persisted in root canals after chemomechanical preparation.
  
  
  CONCLUSIONS
  The RT-qPCR assay provides a sensitive method for the identification of active E. faecalis from endodontic samples. Furthermore, the rRNA-based assay indicated that E. faecalis viable cells persisted in treated root canals, suggesting that it may be a useful tool for monitoring microbial load during endodontic treatment.
authors.affiliation: Discipline of Endodontics, Department of Dentistry, School of Dentistry, University of São Paulo, São Paulo, Brazil. Electronic address: erickapinheiro@usp.com.
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: George T
authors.initials: GT
authors.lastname: Candeiro
authors.affiliation: Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
authors.forename: Sílvia R
authors.initials: SR
authors.lastname: Teixeira
authors.affiliation: Discipline of Endodontics, Department of Dentistry, School of Dentistry, University of São Paulo, São Paulo, Brazil.
authors.forename: Regina C
authors.initials: RC
authors.lastname: Shin
authors.affiliation: Discipline of Endodontics, Department of Dentistry, School of Dentistry, University of São Paulo, São Paulo, Brazil.
authors.forename: Laís C
authors.initials: LC
authors.lastname: Prado
authors.affiliation: Discipline of Endodontics, Department of Dentistry, School of Dentistry, University of São Paulo, São Paulo, Brazil.
authors.forename: Giulio
authors.initials: G
authors.lastname: Gavini
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: D004269:DNA, Bacterial / Q000032:analysis; D003786:Dental Pulp Cavity / Q000382:microbiology*; D013293:Enterococcus faecalis / Q000378:metabolism*; D006801:Humans; D012336:RNA, Ribosomal, 16S / Q000032:analysis*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: Endodontic; Enterococcus faecalis; quantitative polymerase chain reaction; ribosomal RNA–based polymerase chain reaction