基于 rRNA 的逆转录 PCR 与基于 rDNA 的 PCR 检测根管感染链球菌的比较。
Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections.
机构信息
Universidade de São Paulo, Faculdade de Odontologia, São Paulo, SP, Brasil.
Universidade de São Paulo, Faculdade de Odontologia, Instituto de Ciências Biomédicas, São Paulo, SP, Brasil.
出版信息
J Appl Oral Sci. 2019 Jul 29;27:e20180256. doi: 10.1590/1678-7757-2018-0256.
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.
目的
基于 rDNA 的方法无法区分活细胞和死细胞。相反，可以通过基于核糖体 RNA（rRNA）的分子方法来评估细菌的生存能力。因此，本研究旨在通过 rRNA 逆转录聚合酶链反应（RT-PCR）检测根管样本中的存活链球菌，与基于 rDNA 的 PCR 检测方法进行比较。
方法
从 32 颗患有原发性牙髓感染的牙齿中获得微生物根管样本，分别在化学机械预备前（S1）、后（S2）和根管内药物去除后（S3）采集。从 RNA 中提取 RNA 和 DNA，并使用 RT 反应从 RNA 合成 cDNA。将 cDNA 和基因组 DNA 用与链球菌 16S rRNA 序列互补的引物进行 PCR。使用 McNemar 检验比较两种检测方法的检测率（P<0.05）。
结果
使用 rRNA 和 rDNA 基于 PCR 的检测方法，分别在 28.12%（9/32）和 37.5%（12/32）的 S1 样本中检测到链球菌。相比之下，仅在 6.25%（2/32）的 S2 样本中通过 rRNA 基于 RT-PCR 检测到链球菌，而在 15.62%（5/32）的样本中通过 rDNA 基于 PCR 检测到链球菌。最后，在 S3 样本中，rRNA 未检测到链球菌，而 rDNA 基于 PCR 仍在 12.5%（4/32）的样本中检测到细菌。rDNA 基于 PCR 的 PCR 阳性反应总数高于 rRNA 基于检测方法（P<0.05）。
结论
rRNA 基于 RT-PCR 检测链球菌的检出率低于 rDNA 基于 PCR，表明后者可能检测到根管样本中链球菌的死细胞。
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