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Published on: June 12, 2013
RNA-based Assay Demonstrated Enterococcus faecalis Metabolic Activity after Chemomechanical Procedures
Ericka T Pinheiro1, George T Candeiro1, Sílvia R Teixeira2
1Discipline of Endodontics, Department of Dentistry, School of Dentistry, University of São Paulo, São Paulo, Brazil.
Introduction:
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.
