title: Quantitative real-time polymerase chain reaction versus culture: a comparison between two methods for the detection and quantification of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis in subgingival plaque samples.
abstract: OBJECTIVE
  The purpose of this investigation was to validate a real-time quantitative polymerase chain reaction (PCR) assay in identifying and quantifying Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis from subgingival plaque samples taken from subjects with different periodontal conditions, when compared with conventional cultural procedures.
  
  
  METHODS
  Ninety-two adult subjects participated in this study, 32 with periodontitis, 30 with gingivitis and 30 healthy. A pooled subgingival sample was obtained from every patient. Culturing procedures were carried out using standard techniques. For real-time PCR analysis, primers were selected from sequences of the LktC (A. actinomycetemcomitans), Arg-gingipain (P. gingivalis) and BspA antigen (T. forsythensis) genes. Contingency tables were constructed to compare the qualitative results, while quantitative data were evaluated by paired t-test.
  
  
  RESULTS
  A. actinomycetemcomitans was the least frequently recovered species with both techniques. Prevalence of P. gingivalis was low in healthy patients, increased in gingivitis and peaked in periodontitis patients. The frequency of detection of T. forsythensis showed marked differences between culture and PCR, although the same tendency of an increase in prevalence from health to gingivitis and to periodontitis was observed with both methods. Contingency tables demonstrated a good level of agreement between PCR and culture procedures for A. actinomycetemcomitans and P. gingivalis, especially in periodontitis patients. P. gingivalis culture counts were significantly higher than those obtained by PCR. The opposite was true for T. forsythensis, and statistically significant higher counts were obtained by PCR for gingivitis and periodontitis patients.
  
  
  CONCLUSIONS
  This study demonstrated a good agreement between the quantitative PCR technology and the culture procedure. The high sensitivity and specificity of the quantitative PCR technology justify its use in epidemiological studies and as an adjunct in clinical diagnosis of periodontal patients.
authors.affiliation: Laboratory of Microbiology, University Complutense, Madrid 28040, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Lau
authors.forename: Mariano
authors.initials: M
authors.lastname: Sanz
authors.forename: David
authors.initials: D
authors.lastname: Herrera
authors.forename: Juan Manuel
authors.initials: JM
authors.lastname: Morillo
authors.forename: Conchita
authors.initials: C
authors.lastname: Martín
authors.forename: Augusto
authors.initials: A
authors.lastname: Silva
mesh_terms: D000328:Adult; D016976:Aggregatibacter actinomycetemcomitans / Q000302:isolation & purification*; D001439:Bacteroides / Q000302:isolation & purification*; D015169:Colony Count, Microbial / Q000706:statistics & numerical data; D015342:DNA Probes; D003773:Dental Plaque / Q000382:microbiology*; D005260:Female; D005891:Gingivitis / Q000382:microbiology; D006801:Humans; D008297:Male; D008875:Middle Aged; D010518:Periodontitis / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D016966:Porphyromonas gingivalis / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes