In this study, the distribution of P. gingivalis kgp gene polymorphism in subgingival plaque of patients with diabetic periodontitis was observed by cross-sectional study. The Ethical Committee approved the study of the Institute of Dental Disease Control and Prevention of Xuhui District [Shanghai and Xufang Colombian (2020) No.1]. Each participant read and signed the informed consent before sampling.
2.1 Sample Collection
Subgingival plaque samples were collected from patients with diabetic periodontitis treated in the Department of Periodontal Medicine of the Institute of Dental Control in Xuhui District from January 2020 to November 2020, and 49 subgingival plaque samples were obtained, which were divided into the diabetic periodontitis group (21 cases) and the non-diabetic periodontitis group (28 cases). The dental plaque samples of 11 healthy periodontal subjects were analyzed.
2.1.1 Sample information
Samples were taken from 49 periodontitis patients aged 27-68 years in the Department of Periodontology, Xuhui District, Shanghai, China.
(1) Inclusion criteria
a. Diabetic periodontitis group: extensive periodontitis, with the diagnostic criteria defined by the European Federation of Periodontal Diseases and the American Society of Periodontal Diseases ; ≥ 40 years old; complete residual teeth I > 15; no smoking or smoking cessation for more than one year; patients who were diagnosed with type 2 diabetes for more than two years, or whose general health did not exclude individual abnormal blood lipid indicators, but were not diagnosed with metabolic syndrome; non-communicable diseases such as active tuberculosis; sign informed consent.
b. Non-diabetic periodontitis group: extensive periodontitis, whose diagnostic criteria were defined by the European Federation of Periodontal Diseases and the American Society of Periodontal Diseases; ≥ 40 years old; complete residual teeth I > 15; no smoking or smoking cessation for more than one year; hbAlc (glycosylated hemoglobin ) < 7 %; non-communicable diseases such as active tuberculosis ; sign informed consent.
c. Inclusion criteria for periodontal health group: negative bleeding on probing; probing depth < 3mm; no attachment loss; no imaging bone loss; hbAlc < 7 %; non-communicable diseases such as active tuberculosis; sign informed consent.
(2) Exclusion criteria
A history of periodontal system treatment within one year; continuous administration of antibiotics for more than one week within six months before the examination; having multiple bad restorations in the mouth affecting the examiner; women pregnancy or lactation
2.1.2 Sampling process
One mesiobuccal site of anterior and posterior teeth with the most obvious gingival swelling was selected, and the subgingival plaque was collected to avoid adjacent sites of missing teeth and subgingival sites with prosthesis. After aseptic and moisturizing teeth, the subgingival plaque of the tested tooth was taken with a sterile subgingival scraper, suspended in 1 mL PBS buffer, and frozen at -20 °C for DNA extraction. At the same time, periodontal doctors used periodontal probes to record periodontal parameters at six sites of the tested tooth, including plaque index (PI), probing bleeding (BOP), probing depth (PD), gingival retraction (GR), and clinical attachment loss CAL).
2.2 DNA extraction
DNA was extracted from 49 clinical samples by a low-dose bacterial genomic DNA extraction kit (Kaijie Biotechnology (Shanghai) Co., Ltd.), and the obtained DNA was frozen at -20 °C.
2.3 PCR identification of P. gingivalis 16S rRNA gene
The 16S rRNA primers of P. gingivalis
[18]
were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (the primer sequences are shown in Table 1). The polymerase chain reaction amplification system: Ex Hot Start Version 25 μL (Shanghai Sangon Bioengineering Technology Service Co., Ltd.); 2 μL each primer (10 μM); DNA template 2μL, add ddH2O to 50μL. The reaction conditions were as follows: pre-denaturation at 94 °C for 10 min, denaturation at 94 °C for 60 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, 35 cycles, extension at 72 °C for 5 min. In the amplification reaction, the standard strains P. gingivalis W83 and ATCC 33277 were used as a positive control, and the same volume of sterile double distilled water was used as blank control. Take 5μL polymerase chain reaction mixture 1.2 % agarose gel electrophoresis, gel imaging analyzer imaging analysis (electrophoresis Figure 1).
2.4 kgp gene catalytic domain primer specific PCR
The specific primers for the catalytic domain of the kgp gene were designed by reference to Beikler
[14]
(see Table 1 for primer sequences and Figure 2 for electrophoresis diagrams). The polymerase chain reaction amplification system: Ex Hot Start Version 25 μL (Shanghai Sangon Bioengineering Technology Service Co., Ltd.); 2 μL each primer (10 μM); DNA template 2μL, add ddH2O to 50μL. The reaction conditions were as follows: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 45 s, 30 cycles, extension at 72 °C for 10 min. In the amplification reaction, the standard strains P. gingivalis W83 and ATCC 33277 were used as a positive control, and the same volume of sterile double distilled water was used as blank control. Take 5μL polymerase chain reaction mixture 1 % agarose gel electrophoresis, gel imaging analyzer imaging analysis.
Table 1
Specific primer sequence of P. gingivalis 16S rRNA and kgp catalytic domain
primer
|
sequence
|
size/bp
|
P. gingivalis
16SrRNA
|
F 5’- TGTAGATGACTGATGGTGAAAACC-3’
|
197
|
R 5’-ACGTCATCCACACCTTCCTC-3'
|
kgp cd
|
F 5’-GAACTGACGAACATCATTG-3’
|
890
|
R 5’-GCTGGCATTAGCAACACCTG-3’
|
2.5 Digestion of Catalytic Domain Fragment of kgp Gene
Restriction endonuclease Mse I was designed by Beikler
[14]
(see Table 2 for enzyme sites). The PCR products of the catalytic domain of the kgp gene were directly digested by Mse I and detected by 1.2 % agarose gel electrophoresis (see table 3 for typing basis and test results and figure 3 for electrophoresis chart). 50 μL enzyme digestion system: 1 μL of MseI (Shanghai Development Laboratory Reagent Co., Ltd.); 10xNEBuffer (Shanghai Sangon Bioengineering Technology Service Co., Ltd.) 5 μL; polymerase chain reaction product 1μg, add ddH2O to 50μL. The reaction conditions were as follows: enzyme digestion at 37 °C for 15 min. In the enzyme digestion reaction, the standard strains P. gingivalis W83 and ATCC 33277 were used as the positive control, and the same volume of sterile double distilled water was used as the blank control. Take 5 μL enzyme reaction mixture 1.2 % agarose gel electrophoresis, gel imaging analyzer imaging analysis.
Table 2
Mse I restriction site
restriction enzyme cutting site
|
5’-3’
|
135
|
125 CAGCCAACCAT▼TAAATATGGTATGCA
|
423
|
412 AATAACTCGCGT▼TAAGGAGAAAGG
|
Table 3
Classification of two kgp cd genotypes of P. gingivalis
genotype
|
Mse I
|
|
result of survey
|
restriction enzyme cutting site
|
segments
|
segment size/bp
|
Type-Ⅰ
|
+ +
|
3
|
447+288+135
|
Type-Ⅱ
|
- -
|
1
|
890
|
2.6 Statistical methods
According to the experimental data, the detection rate of P. gingivalis, the detection rate of kgp, and the detection rate of the two genotypes of kgp were analyzed. Then we further investigated whether there was a statistical correlation between the detection of different kgp genotypes and diabetes. If all theoretical frequencies in the four tables are greater than or equal to 5, the Pearson χ2 test method is used for analysis. If the number of lattices with theoretical frequency 1 ≤ T < 5 in the table exceeds 1 / 5 of the total lattices, the Fish exact probability method is used for analysis. The data analysis was completed by SAS 8.2 statistical software, and the test level was α = 0.05.