慢性牙髓炎及根尖周炎根管内微生物16SrDNA高通量测序分析

发布时间：2018-08-02 14:35

【摘要】：[目的]牙髓病和根尖周病是由多种微生物混合感染引起的疾病,但各种微生物在感染中的作用地位一直是人们关心的问题。本实验利用高通量测序技术对慢性牙髓炎及慢性根尖周炎根管内微生物进行分析,以探讨牙髓及根尖周感染微生物群落及微生物间的关系,为确定疾病的诊断和鉴别标准及根管治疗过程中的感染控制提供实验基础。[方法]1收集临床诊断为慢性牙髓炎、慢性无瘘型根尖周炎、慢性有瘘型根尖周炎的牙髓组织样本共21例,提取样本DNA,进行两步PCR扩增,实时荧光定量后,采用高通量测序技术进行测序,对所得序列结果进行分析。2对测序所得的有效序列进一步优化,得到优化序列,提取非重复序列,将序列按照97%的相似度进行聚类,得到操作分类单元(Operational Taxonomic Uint,OTU)代表序列,与Silva 119数据库进行比对,找出其最相近且可信度达80%以上的种属信息,得到样本中门、纲、目、科、属、种6个水平的分类单元。3对所得的OTU进行Alpha多样性分析、基于序列相似性聚类OTU分析、物种分类学分析、Beta多样性分析、显著性差异分析、微生物间相关性分析。[结果]1慢性牙髓炎组中2例样本DNA提取失败。对慢性牙髓炎组样本5例、慢性无痿型根尖周炎7例、慢性有瘘型根尖周炎组7例共19例样本的扩增结果进行高通量测序。2测序结果与Silva119数据库进行比对,在97%的相似水平下共获得443320个OTU,归属于19个门,27个纲,53个目,95个科,177个属,159个种。3香农指数图显示测序物种覆盖较好。覆盖率均在99%以上,表明本次测序结果代表了样本的真实情况。稀释曲线图显示本次研究各样本的测序数据量合理。有17种菌属占总样本含量较高,均大于1%,共65.36%,11种菌属在样本中检出率100%,共占总样本含量的44%,其中在全部组比较中差异有统计学意义(P0.05)的菌属有不动杆菌属(Acinetobacter)、奈瑟菌属(Neisseria)、Olsenella菌属、叶杆菌属(Phyllobacterium)、芽孢杆菌属(Bacillus)、链球菌属(Streptococcus)。慢性牙髓炎组与无瘘型慢性根尖周炎组样本比较有7个菌属差异有统计学意义(P0.05),慢性牙髓炎组与有瘘型慢性根尖周炎组样本比较有54个菌属差异有统计学意义(P0.05),无瘘型与有瘘型慢性根尖周炎组样本比较有7个菌属差异有统计学意义(P0.05)。微生物相关性分析,发现慢性牙髓炎组中微生物间有共生关系的有叶杆菌属、不动杆菌属、芽孢杆菌属间等；拮抗关系的有纤毛菌属与叶杆菌属、不动杆菌属、芽孢杆菌属等。无瘘型慢性根尖周炎组中微生物间有共生关系的有不动杆菌属、乳杆菌属、叶杆菌属、芽孢杆菌属间等；拮抗关系的有放线菌属与戴阿利斯特杆菌属等。有瘘型慢性根尖周炎组中微生物间有共生关系的叶杆菌属、不动杆菌属、芽孢杆菌属间等；拮抗关系的有Phocaeicola菌属与普雷沃菌属、乳杆菌属等。[结论]采用高通量测序分析慢性牙髓炎及根尖周炎根管内微生物物种覆盖较好,慢性牙髓炎组、无瘘型慢性根尖周炎组、有瘘型慢性根尖周炎组3组间的微生物组成多样性不同,慢性牙髓炎组、无瘘型慢性根尖周炎组、有瘘型慢性根尖周炎组各组内的微生物间存在着共生和拮抗关系。
[Abstract]:[Objective] dental pulp disease and periapical disease are diseases caused by mixed infection of a variety of microorganisms, but the role of various microbes in infection has always been a concern. This experiment used high throughput sequencing technology to analyze the microbes in the root canal of chronic pulpitis and chronic periapical periodontitis, in order to explore the micro infection of the pulp and periapical periodontitis. The relationship between biological communities and microbes provides an experimental basis for determining the diagnosis and identification of disease and the control of infection in root canal therapy. [methods]1 collected 21 cases of clinical diagnosis as chronic pulpitis, chronic fistula type periapical periodontitis, chronic fistula type periapical periodontitis, and extracted samples of DNA for PCR expansion. After real-time fluorescence quantitative, high throughput sequencing technology was used to sequence the results, and the results of the sequence were analyzed by.2, the effective sequence was further optimized, the optimized sequence was obtained, the non repeated sequence was extracted, and the sequence was clustered according to the similarity of 97%, and the representative sequence of Operational Taxonomic Uint (OTU) was obtained. Compared with the Silva 119 database, the most close and more than 80% of the species information were found, and the 6 level taxa,.3, were used to analyze the Alpha diversity of the OTU, based on the sequence similarity cluster OTU analysis, the taxonomic analysis, the Beta diversity analysis, and the significant difference. Analysis, correlation analysis between microbes. [results]1 chronic pulpitis group with 2 samples of DNA extraction failed. 5 cases of chronic pulpitis group, 7 cases of chronic flaccid periapical periodontitis, 7 cases of chronic fistula type periapical periodontitis group, 19 cases were amplified by high flux sequencing.2 sequencing, compared with Silva119 database, the similarity in 97% was similar. At the level of 443320 OTU, belonging to 19 doors, 27 classes, 53 orders, 95 families, 177 genera, 159 species of.3 Shannon index map showed that the sequence of sequencing species was covered better. The coverage rate was above 99%, indicating that the sequencing results represented the true situation of the samples. The total sample content was higher than 1%, a total of more than 1%, a total of 65.36%. The detection rate of 11 species in the genus was 100%, accounting for 44% of the total sample content. Among all the groups, there were statistically significant differences (P0.05) with Acinetobacter (Acinetobacter), Neisseria (Neisseria), Olsenella, Phyllobacterium, and spore. Bacteria (Bacillus), Streptococcus (Streptococcus), chronic pulpitis group and fistula type chronic periapical periodontitis group samples compared with 7 bacteria differences have statistical significance (P0.05), chronic pulpitis group and chronic periapical periodontitis samples with fistula type, there are 54 differences in statistical significance (P0.05), fistula type and fistula type chronic periapical periodontitis There were 7 bacteria differences in the samples of the inflammatory group (P0.05). The microbial correlation analysis showed that there were symbiotic bacteria, Acinetobacter, and Bacillus in the chronic pulpitis group, and the antagonistic relationship was ciliated and leaf bacillus, Acinetobacter, bacillus and so on. Acinetobacter, lactobacilli, Lactobacillus, and Bacillus sp. among the microbes in the periapical periodontitis group; antagonistic relation of actinomycetes and Day Alister. Phocaeicola bacteria and pervo, Lactobacillus and so on. [Conclusion] high throughput sequencing analysis of chronic pulpitis and periapical periodontitis has better microbial species coverage in the root canal, chronic pulpitis, fistula type chronic periapical periodontitis, and chronic periapical periodontitis with fistula of 3 groups with different microbial diversity and chronic teeth. There were symbiotic and antagonistic relationships among the microorganisms in the pulpitis group, non-fistula chronic periapical periodontitis group and fistula chronic periapical periodontitis group.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R781.3

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