Chuba P J, Pelz K, Krekeler G, de Isele T S, Göbel U
Institut für Medizinische Mikrobiologie und Hygiene der Universität Freiburg, FRG.
J Gen Microbiol. 1988 Jul;134(7):1931-8. doi: 10.1099/00221287-134-7-1931.
Analysis of the subgingival microflora has recently implicated Actinobacillus (Haemophilus) actinomycetemcomitans and several black Bacteroides species in the aetiology of juvenile, adult and rapidly progressing periodontitis. Rapid bacteriological diagnosis has been hampered by the slow growth and fastidious nature of these bacteria. To construct diagnostic probes, dideoxy sequencing of the 16S rRNA molecules from A. (H.) actinomycetemcomitans, Haemophilus aphrophilus, Bacteroides gingivalis, Bacteroides intermedius subgroup II, Bacteroides asaccharolyticus and several closely related species was performed. Next, oligodeoxynucleotides, complementary to defined regions of the 16S rRNA exhibiting considerable evolutionary divergence, were synthesized for use as molecular probes. In a dot-blot hybridization assay, all strains from each of the species for which probes were constructed were correctly identified, with a detection limit of less than 5 x 10(3) organisms. No cross-hybridization to closely related species (except for H. aphrophilus and Haemophilus paraphrophilus) or contaminating bacteria was observed. Using a modified DNA/RNA hybridization technique, the detection could be performed in less than 12 h, as compared to 2-3 weeks using conventional bacteriological procedures.
对龈下微生物区系的分析最近表明，伴放线放线杆菌（嗜沫嗜血杆菌）以及几种黑色拟杆菌属菌种与青少年牙周炎、成人牙周炎和快速进展性牙周炎的病因有关。这些细菌生长缓慢且苛求的特性阻碍了快速细菌学诊断。为构建诊断探针，对来自伴放线放线杆菌（嗜沫嗜血杆菌）、嗜二氧化碳嗜纤维菌、牙龈拟杆菌、中间拟杆菌II亚群、解糖胨拟杆菌以及几种密切相关菌种的16S rRNA分子进行了双脱氧测序。接下来，合成了与16S rRNA特定区域互补的寡脱氧核苷酸，这些区域表现出相当大的进化差异，用作分子探针。在斑点杂交试验中，构建了探针的每个菌种的所有菌株都被正确鉴定，检测限低于5×10³个生物体。未观察到与密切相关菌种（除嗜二氧化碳嗜纤维菌和副嗜二氧化碳嗜纤维菌外）或污染细菌的交叉杂交。使用改良的DNA/RNA杂交技术，检测可在不到12小时内完成，而使用传统细菌学方法则需要2至3周。
