Manz W, Amann R, Szewzyk R, Szewzyk U, Stenström T A, Hutzler P, Schleifer K H
Lehrstuhl für Mikrobiologie, Technische Universität München, Germany.
Microbiology (Reading). 1995 Jan;141 ( Pt 1):29-39. doi: 10.1099/00221287-141-1-29.
Bacteria of the family Legionellaceae form a monophyletic group within the gamma-subclass of Proteobacteria. Based on comparative sequence analysis we constructed two oligonucleotide probes complementary to regions of 16S rRNA characteristic for Legionellaceae. Probe specificities were tested by whole-cell or dot-blot hybridization against 14 serogroups of Legionella pneumophila, 22 different Legionella spp. and 72 non-legionellae reference strains. Using optimized conditions both probes hybridized to all tested strains of L. pneumophila. Probes LEG226 and LEG705 hybridized to 71% and 90% of the Legionella species tested, respectively. With the exception of Methylomonas alba none of the non-target strains showed complete sequence homology within the target molecule. In a preliminary evaluation the results of classical techniques employing selective media, immunofluorescence and the probe assay were in good accordance for routine environmental and clinical isolates. L. pneumophila suspended in drinking water at approximately 10(3)-10(4) c.f.u. ml-1 could be rapidly detected by a combination of membrane filtration on polycarbonate filters and whole-cell hybridization. Even after incubation for 1 year a proportion of the released cells was still detectable. In situ hybridization also facilitated visualization of Legionella spp, cells in model biofilms. A combination of in situ hybridization and confocal laser scanning microscopy (CLSM) was used to analyse the three-dimensional arrangement of L. pneumophila within cells of the ciliated protozoan Tetrahymena pyriformis. Whole-cell probing with 16S rRNA-targeted oligonucleotides could, in the future, complement established techniques like immunofluorescence and PCR in ecological and epidemiological studies of Legionellaceae.
军团菌科细菌在变形菌纲γ亚纲中形成一个单系类群。基于比较序列分析，我们构建了两个与军团菌科16S rRNA特征区域互补的寡核苷酸探针。通过全细胞或斑点杂交，针对嗜肺军团菌的14个血清群、22种不同的军团菌属细菌以及72株非军团菌参考菌株测试了探针的特异性。在优化条件下，两种探针均能与所有测试的嗜肺军团菌菌株杂交。探针LEG226和LEG705分别能与71%和90%的测试军团菌属细菌杂交。除白甲基单胞菌外，非靶标菌株在靶标分子内均未显示出完全的序列同源性。在初步评估中，采用选择性培养基、免疫荧光和探针检测等经典技术的结果与常规环境和临床分离株的结果高度一致。通过聚碳酸酯滤膜过滤和全细胞杂交相结合的方法，可快速检测出悬浮在饮用水中浓度约为10³ - 10⁴ c.f.u. ml⁻¹的嗜肺军团菌。即使在培养1年后，仍有一部分释放的细胞可被检测到。原位杂交还便于观察模型生物膜中的军团菌属细菌细胞。原位杂交与共聚焦激光扫描显微镜（CLSM）相结合，用于分析嗜肺军团菌在梨形四膜虫纤毛原生动物细胞内的三维排列。未来，用靶向16S rRNA的寡核苷酸进行全细胞探测，可在军团菌科的生态学和流行病学研究中补充免疫荧光和PCR等现有技术。
