The Correlation Analysis Of The Rag Locus Genotypes Of Porphyromonas Gingivalis And Chronic Periodontitis

ObjectiveThe aim of the present studies was to detect the disposition of Porphyromonas gingivalis(P. gingivalis) of four different rag locus genotypes, and to detect the expression of rag-1 in RNA level in different periodontal inflamed conditions by polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR). We discussed the disposition regularity of the rag locus genotypes of Porphyromonas gingivalis, and the expression regularity of rag-1 in RNA level in different periodontal inflamed conditions.Methods1. Study subjectsA total of 50 subjects were enrolled in the studies who were selected from the periodontal department of School of Stomatology, China Medical University. Subjects were excluded from the studies if any of the following conditions applied: systemic disease, pregnancy, professional tooth cleaning, periodontal therapy during 1 year 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 conditionWe selected three lesion statuses in chronic periodontitis subjects. Clinical parameters included Probing Depth(PD),clinical attachment loss(CAL)and the loss of alveolar bone by X-ray. All the clinical parameters were measured and recorded by a single examiner. 3. Samples collectionSubgingival plaque samples were obtained with sterile toothpicks into 1ml of PBS for DNA and RNA isolation.4. Samples groupingThe samples of 50 subjects were divide into two groups ,which were light group and moderately-severely group according to the clinical parameters.5. Detection of P. gingivalisGenomic DNA were extracted by phenol and chloroform from plaque and thallus, then amplified with 16S rDNA species-specific primer. The PCR products were subjected to agarose gel electrophoresis on 1.5% agarose gel.6. Detection of the four rag genotypesGenomic DNA of P. gingivalis positive samples were amplified with four different species-specific primers of the four rag genotypes. The PCR products were subjected to agarose gel electrophoresis on 1.5% agarose gel.7. RT-PCR reaction of rag-1 positive samplesThe total RNA of rag-1 positive samples were extracted by the bacteria RNAOUT kit, then adjust them to the same concentration. RT-PCR reaction was carried out by the TaKaRa RNA PCR Kit(AMV)Ver.3.0. P. gingivalis 16S rDNA was selected as the intra-control.8. Statistical analysisWe used SPSS 11.0 statistical software. Chi-squared analysis,t-test and rank correlation test were employed to the studies. Significance level: P<0.05 is considered statistically significant, P<0.01 is considered highly statistically significant.Results1. The detection rate of P. gingivalis was 70.6%, and that of the light group and moderately-severely group were 74.4%and 66.7%, respectively. There were not significant differences between them.2. The detection rates of the four rag genotypes were 60.4%,23.6%,44.3% and 15.1%, respectively. The detection rate of rag-1 was the highest , followed by rag-3, both were higher than those of rag-2 and rag-4 statistically(P<0.01 or P< 0.05). But there were not significant differences between rag-2 and rag-4(P>0.05). Besides, we also detect one sample which did not belong to any of the four genotypes. The allied detection rate of the rag-1 and rag-3 was 19.8%, which was higher than those of any two rag genotypes statistically(P < 0.05).The detection rate of rag-1 from moderately-severely group was higher than that of light group statistically(P<0.05). The allied detection rate of rag-1,3 from moderately and severely group was also higher than that of light group statistically(P<0.05).3. The RNA expression of rag-1 in the light group,moderately group,severely group presented elevated,reduced and elevated. There was positive correlation between the clinical periodontal condition and the light group and severely group. But There was negative correlation between the clinical periodontal condition and moderately group.Conclusions1. The detection rate of rag-1 was the highest of the four rag genotypes,followed by rag-3, indicated that rag-1 and rag-3 may be associated with the chronic periodontitis of Chinese people .The detection rate of rag-1 in moderately and severely group was higer than that in light group, indicated P. gingivalis of rag-1 genotype may be more associated with the moderately and severely chronic periodontitis.2. The RNA expression of rag-1 in light group,moderately group,severely group presented eleduced and elevated, indicated that RNA expression of rag-1 may be changed with the devated,rifferent clinical periodontal inflamed condition.