The Relationship Between The Helicobacter Pylori In Dental Plaque And Gastric Mucosa In Patients With Gastric Disease

The relationship between H.pylori and the disease of gastroenterology have received considerable attention since H.pylori was separated from the gastric biopsy samples in the gastritis patients by Warren and Marshall in 1983. It is confirmed that H.pylori is highly relevant to chronic gastritis, peptic ulcer, gastric carcinoma, gastric MALT lymphoma and other disease.Oral cavity is the entrance of the upper digestive tract. Recently, Some researchers have found that H.pylori can live in oral cavity. Further more, it's highly detectable in oral cavity of dyspepsia patients. Hence, the oral cavity is considered as one of the habitats of H.pylori. And the H.pylori infections in oral cavity is relevant to that in stomach.However, other researchers found that H.pylori is not highly detectable in oral cavity. They think that the temporary existence of H.pylori in the oral cavity is due to the flowing back of the gastric juice.In this research, the DNA of H.pylori was extracted from the oral cavity and gastric biopsy of gastric disease. A polymerase chain reaction (PCR) assay with oligonucleotide primers homologous to a portion of the urease C, cytotoxin associated gene A and vacuole toxin associated gene A of H.pylori was used to detect H.pylori in dental plaque and gastric biopsy samples. The objective of this research is to find out the infection of H.pylori in oral cavity of gastric disease patients, and to analyze and compare the genotype of H.pylori in oral cavity and gastric biopsy samples with PCR-SSCP technique, and to try to find out the correlation between the Helicobacter pylori in dental plaque and gastric mucosa.Materials and methods1. The source of H.pylori strain: All the 141patients were out patients of division of gastroenterology in our hospital. 72 is male and 69 is female with the age from 18 to 69 years. We extracted samples from dental plaque and gastric mucosa of the patients who were infected with H.pylori for investigation.2. Preparation of H.pylori Genomic DNA: Methods of direct splitting and genomic DNA purification kit were used for preparation and purification of H.pylori genomic DNA in dental plaque and gastric biopsy.3.PCR amplification of VacA, UreC and CagA: The oligonucleotide primers were used to amplify PCR product: VacA gene (forward) GTC AGC ATC ACA CCG CAA C, (backward) CTG CTT GAA TGC GCC AAA C; UreC gene (forward) GGA TAA GCT TTT AGG GGT GTT AGG GG, (backward) GCT TAC TTT CTA ACA CTA ACG CGC; CagA gene (forward)AAT ACA CCA ACG CCT CCA AG, (backward) TTG TTG CCG CTT TTG CTC TC. PCR was performed in an automated thermal cycler in a final volume of 100 ul, containing 20pmol VacA gene, 50pmol UreC gene, 10pmol CagA gene, 2.5mmol/l MgCL₂, 3U Ex-Taq polymerase, 10ul 10×PCR buffer, 0.25 mmol/l dNTP, 20ul template. PCR cycling conditions: 94℃ 5min, ×1 cycle; 94℃ 30sec, 50℃ 30sec, 72℃ 1min, ×10 cycles; 72℃ +5 sec/ cycle ×25 cycles; 72℃ 7min, ×1 cycle. The results were identified by 2% agarose electrophoresis.4. SSCP: 8ul PCR amplification products of VavA, UreC and CagA+8ul denaturalization liquid (98% formamide , 10mmol/L EDTA, 0.25% bromophenol blue) → 100℃x10min→ice-water bath→4℃ 110V, 8% polypropylene acrylamide electrophoresis→0.2% silver nitrate 10min →3% sodium carbonate.Results1. The source of H.pylori strain: There were 59 patients were diagnosed as H.pylori infection in total 141 patients. The infectious rate was 41.84%. With gastroscope and histopathologic examination, in all 59 patients, 31 patients were diagnosed as chronic superficial gastritis, 5 patients were diagnosed as chronic atrophic gastritis, 15 patients were diagnosed as peptic ulcer, 5 patients were diagnosed as duodenitis, 1 patient was diagnosed as esophagitis, 1 patient was diagnosed as gastric polyp and 1 was gastric carcinoma.2. PCR: The amplification fragments of VacA, UreC and CagA of H.pylori strain is 190bp, 296bp and 397bp respectively. We detect H.pylori in 59gastric biopsy samples and dental plaque with PCR technique, 37 is positive and 22 is negative in gastric biopsy samp...