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Journal of Clinical Microbiology, March 2000, p. 1085-1093, Vol. 38, No. 3
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Touchdown Enzyme Time Release-PCR for Detection and
Identification of Chlamydia trachomatis, C. pneumoniae, and C. psittaci Using the 16S and
16S-23S Spacer rRNA Genes
Guillermo
Madico,1,2
Thomas C.
Quinn,1,2
Jens
Boman,3 and
Charlotte
A.
Gaydos1,*
Division of Infectious Diseases, The Johns
Hopkins University, Baltimore, Maryland1;
Laboratory of Immunoregulation, National Institute of Allergy
and Infectious Diseases, National Institutes of Health, Bethesda,
Maryland2; and Umeå University, Umeå,
Sweden3
Received 21 July 1999/Returned for modification 22 October
1999/Accepted 8 December 1999
Three touchdown enzyme time release (TETR)-PCR assays were used to
amplify different DNA sequences in the variable regions of the 16S and
16S-23S spacer rRNA genes specific for Chlamydia trachomatis, Chlamydia pneumoniae, and
Chlamydia psittaci as improved tests for sensitive
diagnosis and rapid species differentiation. The TETR-PCR protocol used
60 cycles of amplification, which provided improved analytical
sensitivity (0.004 to 0.063 inclusion-forming unit of
Chlamydia species per PCR). The sensitivity of TETR-PCR with primer set CTR 70-CTR 71 was 96.7%, and the specificity was 99.6%, compared to those of the AMPLICOR PCR for the detection of
C. trachomatis in vaginal swab samples. TETR-PCR for
C. pneumoniae with primer set CPN 90-CPN 91 was 90%
sensitive and 93.3% specific compared with a nested PCR with primer
set CP1/2-CPC/D for clinical respiratory samples. TETR-PCR for C. psittaci with primer set CPS 100-CPS 101 showed substantial
agreement with cell culturing (
, 0.78) for animal tissue samples.
Primer sets were then combined into a single multiplex TETR-PCR test.
The respective 315-, 195-, and 111-bp DNA target products were
precisely amplified when DNA from each of the respective
Chlamydia species or combinations of them was used.
Multiplex chlamydia TETR-PCR correctly identified one strain of each of
the 15 serovars of C. trachomatis, 22 isolates of C. pneumoniae, and 20 isolates of C. psittaci. The
primer sets were specific for each species. No target products were
amplified when DNA from C. pecorum or a variety of other
microorganisms was tested for specificity. TETR-PCR with primers
selected for specific sequences in the 16S and 16S-23S spacer rRNA
genes is a valuable test that could be used either with individual
primers or in a multiplex assay for the identification and
differentiation of Chlamydia species from culture isolates
or for the detection of chlamydiae in clinical samples.
*
Corresponding author. Mailing address: Division of
Infectious Disease, The Johns Hopkins University, 1151 Ross Research
Bldg., 720 Rutland Ave., Baltimore, MD 21205. Phone: (410) 614-0932. Fax: (410) 614-9775. E-mail: cgaydos{at}welch.jhu.edu.
Journal of Clinical Microbiology, March 2000, p. 1085-1093, Vol. 38, No. 3
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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