Detection and differentiation of chlamydiae by fluorescence in situ hybridization
Detection and differentiation of chlamydiae by fluorescence in situ hybridization
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DOI：
10.1128/aem.68.8.4081-4089.2002
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发表时间：
2002-08-01
影响因子：
4.4
通讯作者：
Horn, M
中科院分区：
文献类型：
--
作者：
Poppert, S;Essig, A;Horn, M
衣原体是人类和动物的重要病原体，但衣原体感染的诊断仍然受到检测方法不足的阻碍。使用rRNA靶向寡核苷酸探针的荧光原位杂交（FISH）被广泛用于复杂微生物群落中未培养细菌的研究，最近也被证明是快速检测临床样本中各种细菌病原体的有价值的工具。在这里，我们报告的发展和评估的分层探针集的特异性检测和区分衣原体，特别是C。pneumoniae、C. trachomatis、C.鹦鹉热，以及最近描述的衣原体样细菌，包括新衣原体属和副衣原体属。9个新开发的探针的特异性，成功地证明了原位杂交实验感染阿米巴和HeLa 229细胞，包括共感染肺炎衣原体和沙眼衣原体的HeLa 229细胞。FISH可靠染色衣原体包涵体早在12小时感染后。FISH结合直接荧光抗体染色进一步证实了其敏感性。与以前建立的衣原体检测方法相比，FISH不容易出现假阳性结果，并且可以在一个步骤中检测所有识别的衣原体。
Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and Parachlamydia. The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C pneumoniae and C trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.