慢性牙髓炎及根尖周炎根管內(nèi)微生物16SrDNA高通量測(cè)序分析
發(fā)布時(shí)間：2018-08-02 14:35
【摘要】：[目的]牙髓病和根尖周病是由多種微生物混合感染引起的疾病,但各種微生物在感染中的作用地位一直是人們關(guān)心的問(wèn)題。本實(shí)驗(yàn)利用高通量測(cè)序技術(shù)對(duì)慢性牙髓炎及慢性根尖周炎根管內(nèi)微生物進(jìn)行分析,以探討牙髓及根尖周感染微生物群落及微生物間的關(guān)系,為確定疾病的診斷和鑒別標(biāo)準(zhǔn)及根管治療過(guò)程中的感染控制提供實(shí)驗(yàn)基礎(chǔ)。[方法]1收集臨床診斷為慢性牙髓炎、慢性無(wú)瘺型根尖周炎、慢性有瘺型根尖周炎的牙髓組織樣本共21例,提取樣本DNA,進(jìn)行兩步PCR擴(kuò)增,實(shí)時(shí)熒光定量后,采用高通量測(cè)序技術(shù)進(jìn)行測(cè)序,對(duì)所得序列結(jié)果進(jìn)行分析。2對(duì)測(cè)序所得的有效序列進(jìn)一步優(yōu)化,得到優(yōu)化序列,提取非重復(fù)序列,將序列按照97%的相似度進(jìn)行聚類,得到操作分類單元(Operational Taxonomic Uint,OTU)代表序列,與Silva 119數(shù)據(jù)庫(kù)進(jìn)行比對(duì),找出其最相近且可信度達(dá)80%以上的種屬信息,得到樣本中門、綱、目、科、屬、種6個(gè)水平的分類單元。3對(duì)所得的OTU進(jìn)行Alpha多樣性分析、基于序列相似性聚類OTU分析、物種分類學(xué)分析、Beta多樣性分析、顯著性差異分析、微生物間相關(guān)性分析。[結(jié)果]1慢性牙髓炎組中2例樣本DNA提取失敗。對(duì)慢性牙髓炎組樣本5例、慢性無(wú)痿型根尖周炎7例、慢性有瘺型根尖周炎組7例共19例樣本的擴(kuò)增結(jié)果進(jìn)行高通量測(cè)序。2測(cè)序結(jié)果與Silva119數(shù)據(jù)庫(kù)進(jìn)行比對(duì),在97%的相似水平下共獲得443320個(gè)OTU,歸屬于19個(gè)門,27個(gè)綱,53個(gè)目,95個(gè)科,177個(gè)屬,159個(gè)種。3香農(nóng)指數(shù)圖顯示測(cè)序物種覆蓋較好。覆蓋率均在99%以上,表明本次測(cè)序結(jié)果代表了樣本的真實(shí)情況。稀釋曲線圖顯示本次研究各樣本的測(cè)序數(shù)據(jù)量合理。有17種菌屬占總樣本含量較高,均大于1%,共65.36%,11種菌屬在樣本中檢出率100%,共占總樣本含量的44%,其中在全部組比較中差異有統(tǒng)計(jì)學(xué)意義(P0.05)的菌屬有不動(dòng)桿菌屬(Acinetobacter)、奈瑟菌屬(Neisseria)、Olsenella菌屬、葉桿菌屬(Phyllobacterium)、芽孢桿菌屬(Bacillus)、鏈球菌屬(Streptococcus)。慢性牙髓炎組與無(wú)瘺型慢性根尖周炎組樣本比較有7個(gè)菌屬差異有統(tǒng)計(jì)學(xué)意義(P0.05),慢性牙髓炎組與有瘺型慢性根尖周炎組樣本比較有54個(gè)菌屬差異有統(tǒng)計(jì)學(xué)意義(P0.05),無(wú)瘺型與有瘺型慢性根尖周炎組樣本比較有7個(gè)菌屬差異有統(tǒng)計(jì)學(xué)意義(P0.05)。微生物相關(guān)性分析,發(fā)現(xiàn)慢性牙髓炎組中微生物間有共生關(guān)系的有葉桿菌屬、不動(dòng)桿菌屬、芽孢桿菌屬間等；拮抗關(guān)系的有纖毛菌屬與葉桿菌屬、不動(dòng)桿菌屬、芽孢桿菌屬等。無(wú)瘺型慢性根尖周炎組中微生物間有共生關(guān)系的有不動(dòng)桿菌屬、乳桿菌屬、葉桿菌屬、芽孢桿菌屬間等；拮抗關(guān)系的有放線菌屬與戴阿利斯特桿菌屬等。有瘺型慢性根尖周炎組中微生物間有共生關(guān)系的葉桿菌屬、不動(dòng)桿菌屬、芽孢桿菌屬間等；拮抗關(guān)系的有Phocaeicola菌屬與普雷沃菌屬、乳桿菌屬等。[結(jié)論]采用高通量測(cè)序分析慢性牙髓炎及根尖周炎根管內(nèi)微生物物種覆蓋較好,慢性牙髓炎組、無(wú)瘺型慢性根尖周炎組、有瘺型慢性根尖周炎組3組間的微生物組成多樣性不同,慢性牙髓炎組、無(wú)瘺型慢性根尖周炎組、有瘺型慢性根尖周炎組各組內(nèi)的微生物間存在著共生和拮抗關(guān)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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R781.3
本文編號(hào)：2159752
[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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R781.3
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，本文編號(hào)：2159752
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