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Journal of Clinical Microbiology, February 2002, p. 575-583, Vol. 40, No. 2
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.2.575-583.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Development and Evaluation of Real-Time PCR-Based Fluorescence Assays for Detection of Chlamydia pneumoniae

Maria Lucia C. Tondella,1* Deborah F. Talkington,1 Brian P. Holloway,2 Scott F. Dowell,1 Karyn Cowley,1 Montse Soriano-Gabarro,1 Mitchell S. Elkind,3,4 and Barry S. Fields1

Respiratory Diseases Branch, Division of Bacterial and Mycotic Diseases,1 Scientific Resources Program, National Centers for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333,2 Department of Neurology, College of Physicians and Surgeons, Columbia University,3 Columbia-Presbyterian Medical Center of New York Presbyterian Hospital, New York, New York 100324

Received 17 August 2001/ Returned for modification 10 October 2001/ Accepted 9 November 2001

Chlamydia pneumoniae is an important respiratory pathogen recently associated with atherosclerosis and several other chronic diseases. Detection of C. pneumoniae is inconsistent, and standardized PCR assays are needed. Two real-time PCR assays specific for C. pneumoniae were developed by using the fluorescent dye-labeled TaqMan probe-based system. Oligonucleotide primers and probes were designed to target two variable domains of the ompA gene, VD2 and VD4. The limit of detection for each of the two PCR assays was 0.001 inclusion-forming unit. Thirty-nine C. pneumoniae isolates obtained from widely distributed geographical areas were amplified by the VD2 and VD4 assays, producing the expected 108- and 125-bp amplification products, respectively. None of the C. trachomatis serovars, C. psittaci strains, other organisms, or human DNAs tested were amplified. The amplification results of the newly developed assays were compared to the results of culturing and two nested PCR assays, targeting the 16S rRNA and ompA genes. The assays were compared by testing C. pneumoniae purified elementary bodies, animal tissues, 228 peripheral blood mononuclear cell (PBMC) specimens, and 179 oropharyngeal (OP) swab specimens obtained from ischemic stroke patients or matched controls. The real-time VD4 assay and one nested PCR each detected C. pneumoniae in a single, but different, PBMC specimen. Eleven of 179 OP specimens (6.1%) showed evidence of the presence of C. pneumoniae in one or more tests. The real-time VD4 assay detected the most positive results of the five assays. We believe that this real-time PCR assay offers advantages over nested PCR assays and may improve the detection of C. pneumoniae in clinical specimens.

* Corresponding author. Mailing address: Respiratory Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, MS G-03, 1600 Clifton Rd., N.E., Atlanta, GA 30333. Phone: (404) 639-1239. Fax: (404) 639-4215. E-mail: MTondella{at}CDC.GOV.

Journal of Clinical Microbiology, February 2002, p. 575-583, Vol. 40, No. 2
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.2.575-583.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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Copyright © 2002 by the American Society for Microbiology. All rights reserved.