title: Analysis of viable vs. dead Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis using selective quantitative real-time PCR with propidium monoazide.
abstract: OBJECTIVE
  One of the major disadvantages of DNA-based microbial diagnostics is their inability to differentiate DNA between viable and dead microorganisms, which could be important when studying etiologically relevant pathogens. The aim of this investigation was to optimize a method for the selective detection and quantification of only viable Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis cells by combining quantitative real-time polymerase chain reaction (qPCR) and propidium monoazide (PMA).
  
  
  METHODS
  Three different concentrations of PMA (10, 50 or 100 μm) were added to suspensions of 10(6) (CFU)/mL of viable/dead A. actinomycetemcomitans and P. gingivalis cells. After DNA isolation, qPCR was carried out using specific primers and probes for the tested bacteria. PMA was further tested with different mixtures containing varying ratios of viable and dead cells. The efficacy of PMA to detect viable/dead cells was tested by analysis of variance.
  
  
  RESULTS
  For these specific bacterial pathogens, 100 μm PMA resulted in a significant reduction of qPCR amplification with dead cells (10(6) CFU/mL), while with viable cells no significant inhibition was detected. PMA was also effective in detecting selectively viable cells by qPCR detection, when mixtures of varying ratios of viable and dead bacteria were used.
  
  
  CONCLUSIONS
  This study demonstrated the efficiency of PMA for differentiating viable and dead A. actinomycetemcomitans and P. gingivalis cells. This method of PMA-qPCR may be useful for monitoring new antimicrobial strategies and for assessing the pathogenic potential of A. actinomycetemcomitans and P. gingivalis in different oral conditions when using molecular diagnostic methods.
authors.affiliation: ETEP (Aetiology and Therapy of Periodontal Diseases) Research Group, University Complutense of Madrid, Madrid, Spain.
authors.forename: M C
authors.initials: MC
authors.lastname: Sánchez
authors.forename: M J
authors.initials: MJ
authors.lastname: Marín
authors.forename: E
authors.initials: E
authors.lastname: Figuero
authors.forename: A
authors.initials: A
authors.lastname: Llama-Palacios
authors.forename: D
authors.initials: D
authors.lastname: Herrera
authors.forename: M
authors.initials: M
authors.lastname: Sanz
mesh_terms: D019840:2-Propanol / Q000494:pharmacology; D016976:Aggregatibacter actinomycetemcomitans / Q000145:classification* / Q000187:drug effects; D000891:Anti-Infective Agents, Local / Q000494:pharmacology; D001386:Azides; D058491:Bacterial Load; D001431:Bacteriological Techniques; D004396:Coloring Agents; D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis; D006801:Humans; D050296:Microbial Viability / Q000187:drug effects; D016966:Porphyromonas gingivalis / Q000145:classification* / Q000187:drug effects; D011419:Propidium / Q000031:analogs & derivatives*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D000891:Anti-Infective Agents, Local; D001386:Azides; D004396:Coloring Agents; D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial; C533957:propidium monoazide; D011419:Propidium; D019840:2-Propanol