【作者】 张琛； 侯本祥；

【Author】 Zhang Chen Hou Ben-xiang(Department of Endodontics,School of Stomatology,Capital Medical University,Beijing,100050)

【机构】 首都医科大学口腔医学院牙体牙髓科；

【摘要】 目的:采用16SrDNA克隆测序法揭示持续性根尖周炎根管内细菌构成,检测优势菌,探索未知菌种。方法:通过采集15例持续性根尖周炎根管内的样本,6例无根尖病变的根管治疗不完善作为对照组样本,提取全细菌基因组DNA,选择6例前牙持续性根尖周炎根管样本与6例对照组根管样本DNA采用实时定量PCR比较总细菌量;通用引物扩增1 5个持续性根尖周炎根管内的样本16SrDNA,克隆到大肠杆菌,经蓝白斑筛选和PCR鉴定,挑取含目的片段的克隆子,构建16SrDNA克隆文库。测序鉴定16SrDNA克隆文库中细菌。测序结果与基因库中已知序列比对,鉴定菌种或亲缘关系。结果:15例根管内样本中均有细菌检出,持续性根尖周炎根管内总细菌量显著高于对照组样本,从15个持续性根尖周炎根管样本中共获得获得760个可分析序列,共检测出65个菌种或基因型,平均每样本9.4种菌,检出4种新基因型,19种为未培养菌,占全部克隆总数的13.4%,出现在4个样本以上的优势菌属有Parvimonas sp,Solobacterium sp,Dialister sp,Enterococcussp,Lachnospira sp,Propionibacterium sp,Mogibacterium sp。优势菌种有Parvimonasmicra,Solobacterium moorei,Dialister invisus,Enterococcus faecium,Lachnospiraceaeoral clone MCE7₆0,Filifactor alocis,Olsenella uli,Pseudomonas veronii,Bulleidiamoorei,Fusobacterium nucleatum。多数菌种集中在厚壁菌门（Firmicutes）,变形菌门（Proteobacteria）,拟杆菌门（Bacteroidete）,放线菌门（Actinobacteria）,这4个菌门占据了83%的克隆数,其中厚壁菌门居首位。结论:本研究证明应用1 6SrDNA克隆测序法检测持续性根尖周炎根管内的细菌较传统培养法有明显优势,可以检测不可培养菌和新菌种/基因型。菌群分析更全面,具有更高的敏感性,特异性。持续性根尖周炎根管内存在持续的细菌感染,菌群结构复杂,包括可培养菌和不可培养菌（前者占较大比例）;具有广泛菌种多样性。3.本研究得出持续性根尖周炎根管内的优势菌群有Parvimonasmicra,Solobacterium moorei,Dialister invisus,Enterococcus faecium,Lachnospiraceaeoral clone MCE7₆0,Filifactor alocis,Olsenella uli,Pseudomonas veronii,Bulleidiamoorei,Fusobacterium nucleatum。其中包括可培养和未培养菌。4.本研究检测出的4个新菌基因型,均为非优势菌。更多还原

【Abstract】 OBJECTIV:Persistent/secondary infections of human root canals play an important role in the failure of endodontic treatment.This study used 16S rDNA sequencing to assess microbial diversity in root-filled teeth associated with failed endodontic treatment.METHODS:DNA was extracted from 15 teeth with persistent intraradicular infections,and the 16S rDNA of all present bacteria was amplified by PCR, followed by cloning and sequencing of the 16S rDNA amplicons.RESULTS:All sample extracts were positive for PCR amplification using the universal 16S rDNA gene primers. Negative control reactions yielded no amplicons.Sixty-five phylotypes belonging to seven phyla were identified from 760 clones;a mean of 9.4 phylotypes were detected in each sample(range 3-15).Twenty-eight phylotypes were detected in more than one sample, revealing a high inter-sample variability.Parvimonas micra(60%,9/15),Solobacterium moore(47%,7/15),Dialister invisus(33%,5/15),Enterococcus faecalis(33%5/15), Filifactor alocis(27%,4/15),Fusobacterium nucleatum(27%,4/15)were the predominant bacteria.Nineteen as-yet-uncultivated phylotypes were identified,comprising a substantial proportion of the bacteria in many cases.Four clones with low similarity to known 16S rDNA sequences were extensively sequenced and classified taxonomically as novel representatives of the orders Clostridiales and Lactobacillales.CONCLUSIONS: Persistent intraradicular infections were present in all root-filled teeth associated with failed endodontic treatment.The current observations reveal new candidate endodontic pathogens,including as-yet-uncultivated bacteria and phylotypes that may participate in the mixed infections associated with post-treatment apical periodontitis.更多还原