Study Of RgpB Genetic Polymorphism Of Porphyromonas Gingivalis In Subgingival Plaque

OBJECTIVEThe aim of the present studies was to detect the genetic polymorphism of Arg - gingipainB ( rgpB ) , which is a virulence factor of Porphyromonas gingivalis ( P. gingivalis ) , in the subgingival plaque from the lesion and non - lesion statuses in chronic periodontitis and the healthy status of periodontally healthy subjects. We discussed the role of the various genotypes in the initiation and procession of chronic periodontitis, and It would provide beneficial references with the research of pathogenic mechanism.METHODS1. Study subjects:A total of 62 subjects were enrolled in the studies. The case group included 42 chronic periodontitis patients and the control group included 20 subjects without any periodontal disease and with - age and gender matched with case group. Subjects were excluded from the studies if any of the following conditions applied: smoking or professional tooth cleaning, periodontal therapy or taking any medicine during 3 months preceding to the study. All subjects enrolled into the studies signed the informed consent form.2. Measurement of clinical periodontal parameters:We selected lesion and non - lesion statuses in chronic periodontitis and the healthy status in periodontally healthy subjects. Clinical parameters included bleeding on probing ( BOP ) , clinical attachment loss ( CAL ) , probing depth ( PD) , and tooth movement ( TM) , were measured and recorded by a single ex-aminer.3. Sample collection and bacterial culture;Subgingival plaque samples were obtained with sterile subgingival curettes. For DNA isolation,the plaque samples were pooled in lml of PBS( PH7.4). For isolation of P. gingivalis , the plaque samples were pooled in 1 ml of bacterium refer fluid(TD).4. Detection of P. gingivalis;Genomic DNA were extracted by phenol and chloroform from plaque and thallus, then amplified with 16S rRNA species - specific primer. The PCR products were subjected to agarose gel electrophoresis on 1.5% agarose gel.5. rgpB gene polymorphism analysis:The primers were designed to amplify the gene encoding the catalytic domain of rgpB . The PCR products were digested with restriction endonuclease Mbo II and BsrG I . The products were subjected to agarose gel electrophoresis on 2% agarose gel.6. Statistical analysis;We used SPSS11. 5 statistical software. Chi - squared analysis was employed to the studies. Significance level;P < 0. 05 is considered statistically significant, P < 0.01 is considered highly statistically significant.RESULTS1. By culture method , the detection rate of P. gingivalis from lesion and non - lesion statuses in chronic periodontitis and healthy status in periodontally healthy subjects were 26. 19% , 16. 67% and 0% , respectively. Significant differences were detected between the chronic periodontitis and periodontally healthy subjects ( P < 0.01) , while, no differences were detected between lesion and non - lesion statuses in chronic periodontitis.2. By the method of species - specific 16S rRNA PCR , the detection rate of P. gingivalis from lesion and non - lesion status in chronic periodontitis and healthy status in periodontally healthy subjects were 85. 70% , 76. 19% and 40. 00% , respectively. Significant differences were detected between the chronicperiodontitis and periodontally healthy subjects ( P < 0.01) , while, no differences were detected between lesion and non - lesion statuses in chronic periodontitis.3. Species - specific 16S rRNA PCR was more sensitive than culture method.4. The detection rate of gene rgpB - cd from lesion and non - lesion statuses in chronic periodontitis and healthy status in periodontally healthy subjects were 100. 00% ,90. 63% and 62. 50% , respectively. Differences were detected between the chronic periodontitis and periodontal healthy adults ( P < 0. 05) , while, no differences were detected between lesion and non - lesion statuses in chronic periodontitis.5. rgpB - cd ( + ) and rgpB - cd ( - ) P. gingivalis stains were not detected simultaneously in the same samples.6. In lesion status, the detection rate of I ~ IV rgpB - cd genotypes were 11.11% , 36. 11% , 0% and 52. 79% , respectively. The detection rate of type IV was the highest , followed by H , both were higher than those of type I and IH statistically( P < 0.05 or P < 0.01). But there were not significant differences between type IV and H.7. In non - lesion status, the detection rate of I ~ IV rgpB - cd genotypes were 17. 24% , 75. 86% , 3.45% and 3.45% , respectively. The detection rate of type II was the highest, which higher than those of other types statistically ( P< 0.01) , and no differences were detected among those of type I , HI and IV-8. The occurrences of type I and II were noticeable in periodontally healthy subjects, at rates of 20. 00% and 80.00% respectively.9. The detection rate of type IV in lesion status was higher than that in non -lesion status, while type II lower, statistically( P < 0.01). With regard totype I and HI, no statistically differences were detected between different statuses ( P > 0.05).CONCLUSIONS1. The detection rate of P. gingivalis from chronic periodontitis was higherthan that from periodontally healthy subjects, indicated that P. gingivalis were associated with chronic periodontitis.2. The detection rate oirgpB - cd gene of P. gingivalis from chronic periodontitis was higher than that from periodontally healthy subjects, indicated that the rgpB - cd gene may be associated with the pathogenicity of P. gingivalis .3. There were only one rgpB - cd genotype of P. gingivalis in one subgingi-val plaque sample.4. Multiple rgpB - cd genotypes were detected in sudgingival sample. Type IV P. gingivalis stains were more prevalent in chronic periodontitis, while type II in the other two statuses. It indicated that type IV might be better adapted to environmental challenges, and had stronger pathogenicity.