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Applied and Environmental Microbiology, August 2002, p. 4081-4089, Vol. 68, No. 8
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.8.4081-4089.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization

Sven Poppert,1 Andreas Essig,1 Reinhard Marre,1 Michael Wagner,2 and Matthias Horn2*

Abteilung für medizinische Mikrobiologie und Hygiene, Universität Ulm, Ulm,1 Lehrstuhl für Mikrobiologie, Technische Universität München, Freising, Germany2

Received 26 December 2001/ Accepted 14 May 2002

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.

* Corresponding author. Mailing address: Lehrstuhl für Mikrobiologie, Technische Universität München, Am Hochanger 4, D-85350 Freising, Germany. Phone: 49 8161 7154 59. Fax: 49 8161 7154 75. E-mail: horn{at}microbial-ecology.de.

Applied and Environmental Microbiology, August 2002, p. 4081-4089, Vol. 68, No. 8
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.8.4081-4089.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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