title: Assessment of viable periodontal pathogens by reverse transcription quantitative polymerase chain reaction.
abstract: OBJECTIVE
  Molecular biological methods for the detection of periodontitis-associated bacteria based on DNA amplification have many advantages over classical culture techniques. However, when it comes to assessing immediate therapeutic success, e.g. reduction of viable bacteria, DNA-based polymerase chain reaction is unsuitable because it does not distinguish between live and dead bacteria. Our objective was to establish a simple RNA-based method that is easily set up and allows reliable assessment of the live bacterial load.
  
  
  METHODS
  We compared conventional quantitative real-time PCR (qPCR), propidium monoazide-qPCR and reverse transcription qPCR (RT-qPCR) for the detection of periodontal pathogens after antibiotic treatment in vitro. Applicability was tested using clinical samples of subgingival plaque obtained from patients at different treatment stages.
  
  
  RESULTS
  The bacterial load was remarkably stable over prolonged periods when assessed by conventional qPCR, while both propidium monoazide intercalation as well as cDNA quantitation showed a decline according to decreasing numbers of viable bacteria after antibiotic treatment. Clinical samples of subgingival plaque were directly subjected to DNase I treatment and RT without previous extraction or purification steps. While the results of the DNA- and RNA-based methods are comparable in untreated patients, the classical qPCR frequently detected substantial bacterial load in treated patients where RT-qPCR no longer indicates the presence of those pathogens. The disagreement rates ranged between 4 and 20% in first visit patients and 8-50% in the group of currently treated patients.
  
  
  CONCLUSIONS
  We propose to use RNA-based detection methods to verify the successful eradication of periodontal pathogens.
authors: Lambda (Labor für molekularbiologische DNA Analysen) GmbH, Rainbach, Austria.
authors: M
authors: M
authors: Polonyi
authors: N
authors: N
authors: Prenninger
authors: N B
authors: NB
authors: Arweiler
authors: H
authors: H
authors: Haririan
authors: P
authors: P
authors: Winklehner
authors: S
authors: S
authors: Kierstein
mesh_terms: D016976:Aggregatibacter actinomycetemcomitans / Q000187:drug effects; D000658:Amoxicillin / Q000627:therapeutic use; D000900:Anti-Bacterial Agents / Q000627:therapeutic use; D001386:Azides; D001419:Bacteria / Q000187:drug effects*; D058491:Bacterial Load / Q000187:drug effects; D001439:Bacteroides / Q000187:drug effects; D004269:DNA, Bacterial / Q000032:analysis; D003773:Dental Plaque / Q000382:microbiology; D006801:Humans; D008795:Metronidazole / Q000627:therapeutic use; D050296:Microbial Viability / Q000187:drug effects*; D010518:Periodontitis / Q000188:drug therapy / Q000382:microbiology*; D016966:Porphyromonas gingivalis / Q000187:drug effects; D011419:Propidium / Q000031:analogs & derivatives; D012329:RNA, Bacterial / Q000032:analysis*; D012335:RNA, Ribosomal / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D048373:Treponema denticola / Q000187:drug effects
chemical_list: D000900:Anti-Bacterial Agents; D001386:Azides; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; C533957:propidium monoazide; D008795:Metronidazole; D011419:Propidium; D000658:Amoxicillin
keywords: DNA/RNA; PCR; diagnostics; periodontal; periodontal pathogens; subgingival plaque