OBJECTIVETo establish a 16S rDNA multiplex polymerase chain reaction (PCR) for simultaneously detecting P. gingivalis, A. actinomycetemcomitans and T. denticola in clinical specimens of chronic periodontitis and to study the correlation between different modes of infection and severity of the disease.

METHODSPeriodontal pocket specimens from 152 patients with mild, moderate or advanced chronic periodontitis and gingival sulcus specimens from 30 periodontally healthy individuals were collected and placed in 200 microl lysis buffer. The specimens were incubated in 100 degrees C for 10 min and 10 microl of the supernatant was directly used as PCR template. DNAs from P. gingivalis strain ATCC33277, A. actinomycetemcomitans strain Y4, T. denticola strain FM and E. coli strain DH5alpha were used as positive and negative controls in PCR with all of which were prepared by routing phenol-chloroform method. A multiplex PCR assay, using three sets of primers specific to 16S rDNA genes of the three anaerobes, was developed to detect the specimens. The target amplification fragments from 3 cases of PCR products positive for all the three anaerobes were sequenced after T-A cloning. Chi-square test was applied to analyze the correlation between different coinfection of the three anaerobes and severity of the disease.

RESULTSThe established 16S rDNA multiplex PCR assay was able to detect P. gingivalis, A. actinomycetemcomitans and T. denticola at a minimum of 10, 20 and 20 cells, respectively. In comparison with the reported corresponding sequences, similarities of the nucleotide sequences from the three anaerobes 16S rDNA amplification fragments were as high as 99.45%, 97.08% and 96.59%, respectively. Of the 30 periodontally healthy gingival sulcus specimens, only one (3.3%) positive for P. gingivalis and two (6.7%) for A. actinomycetemcomitans could be identified but all of the rest were negative. In the 152 CP periodontal pocket specimens, 147 cases (96.7%) were positive for P. gingivalis, A. actinomycetemcomitans and/or T. denticola, respectively, and 5 cases (3.3%) were negative for all the three anaerobes. The positive rate of P. gingivalis detection (91.5%, 139/152) was significantly higher than those of A. actinomycetemcomitans (72.4%, 110/152) and T. denticola (80.9%, 123/152) (chi(2) = 7.07, 18.67; P < 0.01). 89.8% of the specimens from patients showed coinfections with two (26.5%) or three anaerobes (63.3%), and the coinfection rates in the specimens from moderate and advanced CP were remarkably higher than that from mild CP (chi(2) = 10.43, P < 0.01).

CONCLUSIONThe 16S rDNA multiplex PCR established in this study showed high sensitivity and specificity, which could be used to detect P. gingivalis, A. actinomycetemcomitans and T. denticola in clinical specimens. CP was an disease caused by multiple pathogenic microbes while the synergistic pathopoiesis of the three microbes was closely related to the severity of the disease.