Clinical Research
RNA-based Assay Demonstrated Enterococcus faecalis Metabolic Activity after Chemomechanical Procedures Ericka T. Pinheiro, PhD,* George T. Candeiro, PhD,* Sılvia R. Teixeira, PhD,† Regina C. Shin, MSc,* Laıs C. Prado, MSc,* Giulio Gavini, PhD,* and M arcia P. A. Mayer, PhD† Abstract 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 RTqPCR 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. (J Endod 2015;-:1–4)
From the *Discipline of Endodontics, Department of Dentistry, School of Dentistry and †Department of Microbiology, Institute of Biomedical Sciences, University of S~ao Paulo, S~ao Paulo, Brazil. Address requests for reprints to Dr Ericka T. Pinheiro, Department of Dentistry, School of Dentistry, University of S~ao Paulo, Av Lineu Prestes, 2227, S~ao Paulo, SP, 05508-000, Brazil. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.04.020
JOE — Volume -, Number -, - 2015
Key Words Endodontic, Enterococcus faecalis, quantitative polymerase chain reaction, ribosomal RNA–based polymerase chain reaction
T
he occurrence of apical periodontitis in previously treated teeth is usually a consequence of persistent or secondary intraradicular infection (1–8). Nonsurgical retreatment is currently used to reduce bacterial load in most infected root canals. Molecular approaches are choice methods to assess the effectiveness in reducing organisms by endodontic procedures (9–11). Their advantages over culturedependent methods include higher sensitivity and the ability to detect uncultivated bacterial species (12). The detection of species-specific macromolecules such as bacterial DNA has simplified bacterial identification processes compared with culture techniques. Moreover, the use of current molecular methods, including quantitative polymerase chain reaction (qPCR), has enabled bacterial quantification based on DNA copy numbers (13). Notably, 1 limitation of DNA-based methodology is its inability to distinguish between live and dead cells, preventing the assessment of antimicrobial therapies (14, 15). One strategy to overcome this problem is the use of RNA-based detection methods because RNA is considered an indicator of microbial viability (9, 11). These methods involve reverse-transcriptase reaction (RT), which synthesizes complementary DNA (cDNA), followed by qPCR using cDNA as the template for amplification (13). RNA targets may be messenger RNA (mRNA) or ribosomal RNA (rRNA). The use of rRNA as a target allows the detection of all bacteria with a universal bacterial primer pair and/or the detection of certain species or genus by using primers complementary to the species or genus-specific rRNA sequences (16). Furthermore, molecular methods targeting rRNA are very sensitive because rRNA is present at high copy numbers in active cells (17). However, because rRNA levels vary greatly depending on the physiological status of the cell, the results of RNA-based methods reflect the metabolic activity of bacteria in the sample population and not their absolute amount (16). Recently, the use of RT-qPCR has been proposed to improve the identification of viable bacteria in systemic diseases (18) and environmental samples (16, 17). In the latter studies, rRNA-based assays (RT-qPCR assays using rRNA-based primers) were more sensitive than those targeting DNA (rRNA genes, termed as ribosomal DNA [rDNA]). Specifically, rRNA-based assays significantly increased the sensitivity for Enterococcus faecalis detection in environmental samples (16). Considering the high prevalence of E. faecalis in persistent and secondary intraradicular infections detected by DNA-based molecular methods (4, 7, 19–22), we hypothesized that rRNA-based qPCR assays would increase the sensitivity of detection of bacterial species in root canal samples. In addition, the correlation between data obtained with rRNA- and rDNA-based assays would give an estimation of bacteria metabolic activity in the sample population. The aim of the present study was to investigate the sensitivity of rRNA-based qPCR (RT-qPCR) for the identification of active E. faecalis in samples obtained from root canals of teeth undergoing retreatment. The results of the rRNA-based assay will contribute to a better understanding of E. faecalis susceptibility to endodontic procedures.
E. faecalis Metabolic Activity
1
Clinical Research Materials and Methods Patient Selection The study protocol was approved by the Ethical Committee in Research of the University of S~ao Paulo (182/10). The sample size was determined based on previous data (16) (power of 80% and significance level of 5%). Twenty teeth were selected from patients who attended the School of Dentistry at the University of S~ao Paulo for nonsurgical endodontic retreatment. A detailed medical and dental history was obtained from each patient. Patients who received antibiotic treatment 3 months before the study and patients with general disease were excluded from the study. All teeth had been previously root filled and showed radiographic evidence of apical periodontitis. Failure of root canal treatment was determined by clinical and radiographic examinations. All teeth had been root canal treated more than 2 years before the study, and the patients were asymptomatic. Although most teeth were coronally restored, coronal leakage because of defective coronal restorations (3/20), old temporary restorative materials (2/20), or coronally unsealed teeth (7/20) were detected in most of the teeth. Clinical and Sampling Procedures Teeth were isolated with rubber dams, and the operative field was disinfected with 30% H2O2 (v/v) and 2.5% sodium hypochlorite (NaOCl) for 30 seconds each followed by 5% sodium thiosulfate to inactivate the disinfectant agents. Access cavities were prepared with sterile high-speed diamond burs under irrigation with sterile physiological solution. Before entering the pulp chamber, the access cavity was disinfected using the protocol described previously, and a bacteriologic sample was taken with sterile paper points as a control to check the sterility of the disinfected tooth surface. Control samples were placed in cryotubes containing phosphate-buffered saline solution, which was inoculated onto m-Enterococcus agar (Oxoid, Hampshire, UK) before storage at 20 C for further DNA extraction. Control samples were tested for Enterococcus spp. growth on agar plates and for the presence of bacterial DNA determined by polymerase chain reaction (PCR). Two of 20 patients were excluded from further participation in the study, because control samples were positive. Root fillings were removed using Gates-Glidden drills (Dentsply Maillefer, Ballaigues, Switzerland) and endodontic files without chemical solvents. Irrigation with sterile saline solution was performed to remove any remaining materials and to moisten the canal before sample collection. For microbial sampling, a #15 Hedstr€om file was introduced in the full length of the canal as determined by the apex locator (Propex II, Dentsply Maillefer), and a filing motion was applied. Next, 3 sterile paper points were consecutively placed on the same root canal level. The file, devoid of the handle, and the paper points were transferred aseptically to cryotubes, which were immediately transferred to the laboratory in an ice bath and submitted to nucleic acid extraction. Chemomechanical preparation was performed using the MTwo rotary system (VDW, Munich, Germany) and copious irrigation with 1% NaOCl. The working length was set at 1 mm short of the foramen as determined by an electronic apex locator. Canals were irrigated with 3 mL 1% NaOCl after changing each file. After the root canal preparation was finished, the canal was irrigated with 3 mL 17% EDTA for 3 minutes followed by 2 mL 1% NaOCl and 2 mL 5% sodium thiosulfate to inactivate the NaOCl solution. Postinstrumentation samples were obtained as described previously. RNA and DNA Extraction Total nucleic acids were extracted using the MasterPure Complete DNA and RNA Purification Kit (Epicentre Technologies, Madison, WI) following the manufacturer’s protocol with an additional bead2
Pinheiro et al.
beating step (17). Briefly, after centrifugation at 10,000g for 10 minutes, supernatants were discarded, and pellets were resuspended in solution containing 450 mL tissue and cell lysis solution. Three hundred milligrams of glass beads (diameter, 0.1 mm; BioSpec Products, Inc, Bartlesville, OK) were added to the suspension, and the mixture was vortexed vigorously for 60 seconds using a Mini-Beadbeater (BioSpec Products) at a power level of 5.0. After bead beating, 2 mL proteinase K was added to the suspension, which was incubated for 15 minutes at 65 C. After incubation, mixtures were cooled on ice for 5 minutes and added to 225 mL MPC protein precipitation reagent (Epicentre Technologies). After centrifugation at 10,000g for 10 minutes, supernatants were collected and subjected to isopropanol precipitation. Total nucleic acid samples were resuspended in 35 mL Tris-EDTA buffer and divided in 2 vials. One vial comprised the DNA sample. DNA was eliminated from total nucleic acid preparation in the other vial by DNAse treatment following the manufacturer’s protocol (Epicentre Technologies). An additional DNAse treatment was performed using DNase I (Invitrogen, S~ao Paulo, Brazil), and the absence of contaminating DNA in RNA samples was confirmed by PCR. RNA and DNA concentrations were measured using a NanoDrop ND 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). DNA was stored at 20 C until use. RT-PCR assays were performed on the same day as extraction.
RT-PCR The synthesis of cDNA was obtained by reverse transcription using the SuperScript III First-Strand Synthesis System (Invitrogen) following the manufacturer’s instructions. cDNA was synthesized using 8 mL RNA sample, random hexamers, and cDNA synthesis mix in a total volume of 20 mL; cDNA was stored at 20 C until use. qPCR Assays Real-time PCR was performed using 2 mL cDNA or 2 mL total nucleic acids as the template and primers complementary for E. faecalis rDNA sequences. Quantitative PCR assays were performed using the StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, CA). PCR reactions were set up in 96-well plates in a total volume of 22 mL containing 10 mL Power SYBR Green PCR Master Mix (Applied Biosystems) and 100 nmol/L each primer (50 -CGCTTCTTTCCTCCC GAGT-30 and 50 -GCCATGCGGCATAAACTG-30 ) (9). Deionized water was used instead of template DNA as the negative control. Cycling conditions for qPCR reactions were 95 C for 10 minutes and 40 cycles of 95 C for 15 seconds and 60 C for 1 minute. Melting curve analyses were performed from 65 C to 95 C to confirm specificity of the amplified products. Data were analyzed using the Applied Biosystems SDS 7500 software. The standard curve was built using recombinant plasmids containing the 1500 fragment encoding 16S rRNA gene of E. faecalis as previously described (23). Plasmid standard dilutions (from 107 to 10 DNA copies) were run in triplicate, and the limit of quantification was 10 DNA copies/reaction. Correlation coefficient (r2), amplification efficiency (E), and y-intercept values were 0.998, 99.8%, and 35.5, respectively. cDNA and DNA samples were run in triplicate, and the mean values for rRNA and rDNA measurements were used to calculate the total number of 16S rRNA and16S rDNA copies per root canal sample. To estimate E. faecalis metabolic activity, ratios between the number of 16S rRNA copies and 16S rDNA copies were calculated for samples with positive qPCR results using both assays. Data Analyses The McNemar test was used to compare the detection frequency (E. faecalis presence/absence) of RNA and DNA-based qPCR assays. JOE — Volume -, Number -, - 2015
Clinical Research TABLE 1. Enterococcus faecalis Detection rates of Ribosomal RNA (rRNA) and Ribosomal DNA (rDNA)-based Assays S1
Enterococcus faecalis rRNA
P value*
rDNA +
+ 10 3
S2
4 1
1.00
rDNA + 5 1
6 6
P value* 0.125
+, samples with positive quantitative polymerase chain reaction results; , samples with negative quantitative polymerase chain reaction results; S1, samples taken before chemomechanical procedures; S2, samples taken after chemomechanical procedures. *The McNemar test was used to compare the difference between the detection rates of the methods. (P < .05 indicates statistically significant differences.)
The Wilcoxon signed rank test was used to compare the number of rRNA and rDNA copies in both qPCR assays and to assess for differences in RNA/DNA ratios in samples taken before (S1) and after chemomechanical preparation (S2). The Wilcoxon matched pairs test was used to evaluate the reduction in the number of rRNA and rDNA copies from S1 to S2. Differences were considered statistically significant when P was
RNA-based Assay Demonstrated Enterococcus faecalis Metabolic Activity after Chemomechanical Procedures Ericka T. Pinheiro, PhD,* George T. Candeiro, PhD,* Sılvia R. Teixeira, PhD,† Regina C. Shin, MSc,* Laıs C. Prado, MSc,* Giulio Gavini, PhD,* and M arcia P. A. Mayer, PhD† Abstract 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 RTqPCR 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. (J Endod 2015;-:1–4)
From the *Discipline of Endodontics, Department of Dentistry, School of Dentistry and †Department of Microbiology, Institute of Biomedical Sciences, University of S~ao Paulo, S~ao Paulo, Brazil. Address requests for reprints to Dr Ericka T. Pinheiro, Department of Dentistry, School of Dentistry, University of S~ao Paulo, Av Lineu Prestes, 2227, S~ao Paulo, SP, 05508-000, Brazil. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.04.020
JOE — Volume -, Number -, - 2015
Key Words Endodontic, Enterococcus faecalis, quantitative polymerase chain reaction, ribosomal RNA–based polymerase chain reaction
T
he occurrence of apical periodontitis in previously treated teeth is usually a consequence of persistent or secondary intraradicular infection (1–8). Nonsurgical retreatment is currently used to reduce bacterial load in most infected root canals. Molecular approaches are choice methods to assess the effectiveness in reducing organisms by endodontic procedures (9–11). Their advantages over culturedependent methods include higher sensitivity and the ability to detect uncultivated bacterial species (12). The detection of species-specific macromolecules such as bacterial DNA has simplified bacterial identification processes compared with culture techniques. Moreover, the use of current molecular methods, including quantitative polymerase chain reaction (qPCR), has enabled bacterial quantification based on DNA copy numbers (13). Notably, 1 limitation of DNA-based methodology is its inability to distinguish between live and dead cells, preventing the assessment of antimicrobial therapies (14, 15). One strategy to overcome this problem is the use of RNA-based detection methods because RNA is considered an indicator of microbial viability (9, 11). These methods involve reverse-transcriptase reaction (RT), which synthesizes complementary DNA (cDNA), followed by qPCR using cDNA as the template for amplification (13). RNA targets may be messenger RNA (mRNA) or ribosomal RNA (rRNA). The use of rRNA as a target allows the detection of all bacteria with a universal bacterial primer pair and/or the detection of certain species or genus by using primers complementary to the species or genus-specific rRNA sequences (16). Furthermore, molecular methods targeting rRNA are very sensitive because rRNA is present at high copy numbers in active cells (17). However, because rRNA levels vary greatly depending on the physiological status of the cell, the results of RNA-based methods reflect the metabolic activity of bacteria in the sample population and not their absolute amount (16). Recently, the use of RT-qPCR has been proposed to improve the identification of viable bacteria in systemic diseases (18) and environmental samples (16, 17). In the latter studies, rRNA-based assays (RT-qPCR assays using rRNA-based primers) were more sensitive than those targeting DNA (rRNA genes, termed as ribosomal DNA [rDNA]). Specifically, rRNA-based assays significantly increased the sensitivity for Enterococcus faecalis detection in environmental samples (16). Considering the high prevalence of E. faecalis in persistent and secondary intraradicular infections detected by DNA-based molecular methods (4, 7, 19–22), we hypothesized that rRNA-based qPCR assays would increase the sensitivity of detection of bacterial species in root canal samples. In addition, the correlation between data obtained with rRNA- and rDNA-based assays would give an estimation of bacteria metabolic activity in the sample population. The aim of the present study was to investigate the sensitivity of rRNA-based qPCR (RT-qPCR) for the identification of active E. faecalis in samples obtained from root canals of teeth undergoing retreatment. The results of the rRNA-based assay will contribute to a better understanding of E. faecalis susceptibility to endodontic procedures.
E. faecalis Metabolic Activity
1
Clinical Research Materials and Methods Patient Selection The study protocol was approved by the Ethical Committee in Research of the University of S~ao Paulo (182/10). The sample size was determined based on previous data (16) (power of 80% and significance level of 5%). Twenty teeth were selected from patients who attended the School of Dentistry at the University of S~ao Paulo for nonsurgical endodontic retreatment. A detailed medical and dental history was obtained from each patient. Patients who received antibiotic treatment 3 months before the study and patients with general disease were excluded from the study. All teeth had been previously root filled and showed radiographic evidence of apical periodontitis. Failure of root canal treatment was determined by clinical and radiographic examinations. All teeth had been root canal treated more than 2 years before the study, and the patients were asymptomatic. Although most teeth were coronally restored, coronal leakage because of defective coronal restorations (3/20), old temporary restorative materials (2/20), or coronally unsealed teeth (7/20) were detected in most of the teeth. Clinical and Sampling Procedures Teeth were isolated with rubber dams, and the operative field was disinfected with 30% H2O2 (v/v) and 2.5% sodium hypochlorite (NaOCl) for 30 seconds each followed by 5% sodium thiosulfate to inactivate the disinfectant agents. Access cavities were prepared with sterile high-speed diamond burs under irrigation with sterile physiological solution. Before entering the pulp chamber, the access cavity was disinfected using the protocol described previously, and a bacteriologic sample was taken with sterile paper points as a control to check the sterility of the disinfected tooth surface. Control samples were placed in cryotubes containing phosphate-buffered saline solution, which was inoculated onto m-Enterococcus agar (Oxoid, Hampshire, UK) before storage at 20 C for further DNA extraction. Control samples were tested for Enterococcus spp. growth on agar plates and for the presence of bacterial DNA determined by polymerase chain reaction (PCR). Two of 20 patients were excluded from further participation in the study, because control samples were positive. Root fillings were removed using Gates-Glidden drills (Dentsply Maillefer, Ballaigues, Switzerland) and endodontic files without chemical solvents. Irrigation with sterile saline solution was performed to remove any remaining materials and to moisten the canal before sample collection. For microbial sampling, a #15 Hedstr€om file was introduced in the full length of the canal as determined by the apex locator (Propex II, Dentsply Maillefer), and a filing motion was applied. Next, 3 sterile paper points were consecutively placed on the same root canal level. The file, devoid of the handle, and the paper points were transferred aseptically to cryotubes, which were immediately transferred to the laboratory in an ice bath and submitted to nucleic acid extraction. Chemomechanical preparation was performed using the MTwo rotary system (VDW, Munich, Germany) and copious irrigation with 1% NaOCl. The working length was set at 1 mm short of the foramen as determined by an electronic apex locator. Canals were irrigated with 3 mL 1% NaOCl after changing each file. After the root canal preparation was finished, the canal was irrigated with 3 mL 17% EDTA for 3 minutes followed by 2 mL 1% NaOCl and 2 mL 5% sodium thiosulfate to inactivate the NaOCl solution. Postinstrumentation samples were obtained as described previously. RNA and DNA Extraction Total nucleic acids were extracted using the MasterPure Complete DNA and RNA Purification Kit (Epicentre Technologies, Madison, WI) following the manufacturer’s protocol with an additional bead2
Pinheiro et al.
beating step (17). Briefly, after centrifugation at 10,000g for 10 minutes, supernatants were discarded, and pellets were resuspended in solution containing 450 mL tissue and cell lysis solution. Three hundred milligrams of glass beads (diameter, 0.1 mm; BioSpec Products, Inc, Bartlesville, OK) were added to the suspension, and the mixture was vortexed vigorously for 60 seconds using a Mini-Beadbeater (BioSpec Products) at a power level of 5.0. After bead beating, 2 mL proteinase K was added to the suspension, which was incubated for 15 minutes at 65 C. After incubation, mixtures were cooled on ice for 5 minutes and added to 225 mL MPC protein precipitation reagent (Epicentre Technologies). After centrifugation at 10,000g for 10 minutes, supernatants were collected and subjected to isopropanol precipitation. Total nucleic acid samples were resuspended in 35 mL Tris-EDTA buffer and divided in 2 vials. One vial comprised the DNA sample. DNA was eliminated from total nucleic acid preparation in the other vial by DNAse treatment following the manufacturer’s protocol (Epicentre Technologies). An additional DNAse treatment was performed using DNase I (Invitrogen, S~ao Paulo, Brazil), and the absence of contaminating DNA in RNA samples was confirmed by PCR. RNA and DNA concentrations were measured using a NanoDrop ND 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). DNA was stored at 20 C until use. RT-PCR assays were performed on the same day as extraction.
RT-PCR The synthesis of cDNA was obtained by reverse transcription using the SuperScript III First-Strand Synthesis System (Invitrogen) following the manufacturer’s instructions. cDNA was synthesized using 8 mL RNA sample, random hexamers, and cDNA synthesis mix in a total volume of 20 mL; cDNA was stored at 20 C until use. qPCR Assays Real-time PCR was performed using 2 mL cDNA or 2 mL total nucleic acids as the template and primers complementary for E. faecalis rDNA sequences. Quantitative PCR assays were performed using the StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, CA). PCR reactions were set up in 96-well plates in a total volume of 22 mL containing 10 mL Power SYBR Green PCR Master Mix (Applied Biosystems) and 100 nmol/L each primer (50 -CGCTTCTTTCCTCCC GAGT-30 and 50 -GCCATGCGGCATAAACTG-30 ) (9). Deionized water was used instead of template DNA as the negative control. Cycling conditions for qPCR reactions were 95 C for 10 minutes and 40 cycles of 95 C for 15 seconds and 60 C for 1 minute. Melting curve analyses were performed from 65 C to 95 C to confirm specificity of the amplified products. Data were analyzed using the Applied Biosystems SDS 7500 software. The standard curve was built using recombinant plasmids containing the 1500 fragment encoding 16S rRNA gene of E. faecalis as previously described (23). Plasmid standard dilutions (from 107 to 10 DNA copies) were run in triplicate, and the limit of quantification was 10 DNA copies/reaction. Correlation coefficient (r2), amplification efficiency (E), and y-intercept values were 0.998, 99.8%, and 35.5, respectively. cDNA and DNA samples were run in triplicate, and the mean values for rRNA and rDNA measurements were used to calculate the total number of 16S rRNA and16S rDNA copies per root canal sample. To estimate E. faecalis metabolic activity, ratios between the number of 16S rRNA copies and 16S rDNA copies were calculated for samples with positive qPCR results using both assays. Data Analyses The McNemar test was used to compare the detection frequency (E. faecalis presence/absence) of RNA and DNA-based qPCR assays. JOE — Volume -, Number -, - 2015
Clinical Research TABLE 1. Enterococcus faecalis Detection rates of Ribosomal RNA (rRNA) and Ribosomal DNA (rDNA)-based Assays S1
Enterococcus faecalis rRNA
P value*
rDNA +
+ 10 3
S2
4 1
1.00
rDNA + 5 1
6 6
P value* 0.125
+, samples with positive quantitative polymerase chain reaction results; , samples with negative quantitative polymerase chain reaction results; S1, samples taken before chemomechanical procedures; S2, samples taken after chemomechanical procedures. *The McNemar test was used to compare the difference between the detection rates of the methods. (P < .05 indicates statistically significant differences.)
The Wilcoxon signed rank test was used to compare the number of rRNA and rDNA copies in both qPCR assays and to assess for differences in RNA/DNA ratios in samples taken before (S1) and after chemomechanical preparation (S2). The Wilcoxon matched pairs test was used to evaluate the reduction in the number of rRNA and rDNA copies from S1 to S2. Differences were considered statistically significant when P was
