Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections

Abstract Objective The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. Methodology Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar’s test was used to compare the detection rate of both assays (P<0.05). Results Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). Conclusions The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.


Introduction
Molecular assays targeting ribosomal DNA genes (rDNA) have been extensively used in endodontic microbiology studies. [1][2][3][4][5][6][7][8][9] These methods are sensitive, fast, and allow the detection/quantification of endodontic infectious agents. 10 However, one limitation of the rDNA-based method is the inability to distinguish between live and dead cells, which may result in an overestimation of bacterial targets in root canals, especially in post-treatment samples. 11,12 Bacterial viability may thus be assessed by molecular methods based on ribosomal RNA (rRNA).
Ribosomal RNA can be considered an indicator of microbial viability because they degrade more rapidly than rDNA after cell death. 10 Moreover, since the number of ribosomes per cell correlates with bacterial growth rate, rRNA-based methods are considered highly sensitive for detecting active bacterial cells in a community. [13][14][15] Since the microbial community profile of root canals comprises many uncultivated bacterial species, rRNA-based molecular methods can be expected to grow in importance as instruments used to monitor viable bacterial loads during endodontic treatment. However, little evidence exists about bacterial activity in endodontic microbial communities using rRNA-based methods. 3,15 Recently, we have compared the sensitivity of rRNA-based reverse transcription quantitative polymerase chain reaction (RT-qPCR) and rDNA-based qPCR assays for the detection of Enterococcus faecalis in persistent/secondary endodontic infections. 15   pushed against the root canal walls to suspend bacteria into the solution. Five sterile paper points were placed individually inside the root canal for 1 min each to collect the initial bacteria content (S1). Both the paper points and the H-file, without the handle, were transferred to cryotubes containing 300 µL of RNAlater solution (Life Technologies, Carlsbad, CA, USA) and frozen at -80°C. In each case, a single root canal was sampled to confine the microbial evaluation to a single ecological environment.
Root canal preparation was performed with R40 or R50 Reciproc instruments (VDW GmbH, Munich, Germany), depending on the initial diameter of the root canal. The selection of instruments to be used followed the manufacturer's instructions. All instruments were used only once and then discarded.
Each canal received an initial flush with 10 mL of 2.5% NaOCl delivered by a disposable syringe and 30G side-vented endodontic needles (EndoEZE, Ultradent Products Inc., South Jordan, UT, USA); next, the Reciproc instrument was inserted into the cervical third with an in-and-out pecking motion. If more pressure was needed to advance the instrument further into the canal after one cycle of three in-and-out movements, the file was removed, and its flutes were cleaned. The root canal was irrigated again with 10 mL of 2.5% NaOCl, and a new cycle of three in-and-out movements was performed in the middle third, followed by new irrigation. The instrument was inserted up to the working length with a brushing motion against the root canal walls. At the end of the preparation, the canal was irrigated with 10 mL of 2.5% NaOCl, for 40 mL volume in total. The canal was then dried using paper points and flushed with 5 mL of 5% sodium thiosulfate for 1 min. Finally, the root canal was filled with sterile saline, and a post-instrumentation sample (S2) was taken as described above.
Following, the root canal was irrigated with 2.5% NaOCl and 17% EDTA, dried using paper points, and

Results
All control samples were PCR-negative, which indicated no bacterial contamination. Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using the rRNA-and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA, compared with 15.62% (5/32) using rDNA.  rDNA-based method. [13][14][15] However, this study revealed The PCR detection rate in this study was higher than that of RT-PCR. This finding is in contrast with our previous study, in which the rRNA-based molecular assay was more sensitive to detect E. faecalis in root canals. 15 The differences between the two studies may be related to the molecular methods used for nucleic acid detection. In our previous study, the use of RT-qPCR may have allowed the detection of low rRNA levels, leading to an increased detection rate of E. faecalis by that method. Moreover, the discrepancy between the present findings for streptococci when compared to our previous findings for E. faecalis may reflect differences in bacterial susceptibility to endodontic treatment procedures. confirmed previous studies, suggesting that DNA detection may not always be associated with active endodontic infection. Therefore, rRNA-based methods may be more suitable to identify functional bacteria that effectively contribute to persistent endodontic infections.