Quantitative real-time polymerase chain reaction based on single copy gene sequence for detection of periodontal pathogens.
OBJECTIVE
To develop a method for quantification of Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf) from subgingival plaque samples based on TaqMan real-time polymerase chain reaction (PCR) technology.


METHODS
Bacterial cells from these species were obtained after culturing reference strains and were counted microscopically. Cellular suspensions in Tris-EDTA buffer were used for DNA extraction after boiling for 20 min. Primers for PCR were selected from sequences of the LktC (Aa), Arg-gingipain (Pg) and BspA antigen (Tf) genes in order to yield amplicons below 100 bp. TaqMan-based real-time PCR was adjusted to quantify each species separately. Cycle threshold (C(T)) values were calculated for each species according to the initial number of copies. A reliability analysis was carried out using intra-class correlation coefficients (ICCs) with a two-way random effects model.


RESULTS
A high sensitivity and specificity was obtained for the detection of the three bacterial species. The TaqMan real-time PCR technology yielded a good repeatability in the obtained cycle threshold (C(T)) values for each initial number of copies, demonstrating coefficients of variation below 5% for each bacteria. The reproducibility of the technique was also demonstrated by the high ICCs (>0.98; p<0.00001) obtained for each bacteria with and without the addition of subgingival plaque.


CONCLUSIONS
A novel diagnostic method based on TaqMan real-time PCR was developed for the quantification of Aa, Pg and Tf. It has demonstrated good sensitivity and repeatability on pure cultures. Its diagnostic utility should be demonstrated in subgingival plaque samples.
Morillo
Juan Manuel
JM
Laboratory of Microbiology, University Complutense, Madrid 28040, Spain.
Lau
Laura
L
Sanz
Mariano
M
Herrera
David
D
Martín
Conchita
C
Silva
Augusto
A
D016976:Aggregatibacter actinomycetemcomitans / Q000235:genetics / Q000302:isolation & purification*; D001439:Bacteroides / Q000235:genetics / Q000302:isolation & purification*; D015169:Colony Count, Microbial; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D010510:Periodontal Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D016966:Porphyromonas gingivalis / Q000235:genetics / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial