Comprehensive and quantitative profiling of microflora of human apical periodontitis

本研究以16S rRNA基因为基础，采用实时定量聚合酶链式反应、聚合酶链式反应克隆、聚合酶链式反应序列分析等方法，对根管治疗前后根管微生物区系进行了研究。每个受试者都获得知情同意，并对有根尖周病变的单根牙进行调查。在通道打开时，从根管中收集每个牙本质样本。当根尖周病变经化学机械清洗和根管内给药临床愈合后，再取根管牙本质标本。根据16S rRNA基因设计通用引物，采用实时荧光定量聚合酶链式反应检测细菌总DNA。对扩增产物进行克隆和部分测序。然后通过NCBI(国家生物技术信息中心)的网站使用BLAST搜索程序将部分16S rRNA基因序列与GenBank数据库中的序列进行比较。根管治疗后细菌DNA浓度明显低于开放根管时。聚合酶链式反应克隆和序列分析显示，根管治疗后以梭杆菌为主，根管治疗后以假单胞菌、慢生根瘤菌和甲基杆菌为主。这些结果表明，根管治疗后，根管微生物区系发生了明显的变化。

The purpose of the present study was to profile microflora of root canals before and after root canal therapy, using real-time PCR, PCR-cloning, PCR-sequence analyses based on 16S rRNA genes. Informed consent was obtained from each subject and single-rooted teeth with periapical lesions were investigated. Upon access opening, each dentin sample was collected from the root canal. When the periapical lesion healed clinically through chemo-mechanical cleaning and intracanal medication, the root canal dentin sample was obtained again. The quantification of total bacterial DNA was performed by real-time PCR using universal primers based on 16S rRNA genes. PCR products were cloned and partially sequenced. The partial 16S rRNA gene sequences were then compared with those from the GenBank database using the Blast search program through the website of the NCBI (National Center for Biotechnology Information). The concentrations of bacterial DNA after root canal therapy were lower than those upon access opening. The PCR-cloning and PCR-sequence analyses revealed that Fusobacterium were initially predominant, and that Pseudomonas, Bradyrhizobium and Methylobacterium were predominant after root canal therapy. These results indicated that drastic shifts occurred in microflora of root canals by root canal therapy.