DIVERSITY OF CULTIVABLE AND UNCULTIVABLE ORAL SPIROCHETES FROM A PATIENT WITH SEVERE DESTRUCTIVE PERIODONTITIS
DIVERSITY OF CULTIVABLE AND UNCULTIVABLE ORAL SPIROCHETES FROM A PATIENT WITH SEVERE DESTRUCTIVE PERIODONTITIS
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DOI：
10.1128/iai.62.5.1889-1895.1994
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发表时间：
1994-05-01
影响因子：
3.1
通讯作者：
GOBEL, UB
中科院分区：
文献类型：
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作者：
CHOI, BK;PASTER, BJ;GOBEL, UB
为了确定严重牙周炎患者牙龈缝隙中可培养和不可培养螺旋体的遗传多样性，从龈下菌斑样本中提取的DNA和RNA的pcr扩增产物中克隆了部分16S rRNA基因。使用普遍保守的引物，在pcr中扩增了约500个bp。将纯化的PCR产物用质粒载体pUC19克隆到大肠杆菌中。用放射性标记的密螺旋体特异性寡核苷酸探针进行集落杂交筛选得到的克隆文库。然后用标准程序对81个螺旋体克隆的16S rRNA插入片段进行测序。将序列与35种螺旋体的16S rRNA序列进行比较，其中包括4种已知可培养的口腔螺旋体。分析结果显示，来自同一患者的口腔密螺旋体具有意想不到的多样性。当98%或更高的序列相似度作为种级聚类的定义时，发现克隆序列代表23个物种。当相似度为92%时，克隆可分为8个主要类群，其中只有文氏密螺旋体和齿状密螺旋体两个类群有已命名的种，而果皮螺旋体和索氏密螺旋体在所有类群中均未出现。81个螺旋体克隆中有7个被发现含有嵌合16S rRNA序列。用异硫氰酸荧光素标记的寡核苷酸探针原位荧光杂交技术直接鉴定临床样品中靶种的细胞。
To determine the genetic diversity of cultivable and uncultivable spirochetes in the gingival crevice of a patient with severe periodontitis, partial 16S rRNA genes were cloned from PCR-amplified products of DNA and RNA extracted from a subgingival plaque sample. Approximately 500 bp were amplified in PCRs by using universally conserved primers with polylinker tails. Purified PCR products were cloned into Escherichia coli by using the plasmid vector pUC19. The resultant clone library was screened by colony hybridization with a radiolabeled, treponeme-specific oligonucleotide probe. The 16S rRNA inserts of 81 spirochetal clones were then sequenced by standard procedures. Sequences were compared with 16S rRNA sequences of 35 spirochetes, including the four known cultivable oral treponeme species. The analysis revealed an unexpected diversity of oral treponemes from a single patient. When 98% or greater sequence similarity was used as the definition of a species-level cluster, the clone sequences were found to represent 23 species. When 92% similarity was used as the definition, the clones fell into eight major groups, only two of which contained named species, Treponema vincentii and Treponema denticola, while Treponema pectinovorum and Treponema socranskii mere not represented in any cluster. Seven of the 81 spirochetal clones were found to contain chimeric 16S rRNA sequences. In situ fluorescence hybridization with a fluorescein isothiocyanate-labeled oligonucleotide probe specific for one of the new species representing cluster 19 was used to identify cells of the target species directly in clinical samples.