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

Abstract

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 (kappa, 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.

FIG. 1

Alignment of the target sequences of primer set CTR 70/71 in the 16S rRNA gene of C. trachomatis with the 16S rRNA genes of other vaginal pathogens. A dot indicates the same base, and a letter indicates a base different from that in C. trachomatis.

FIG. 2

Alignment of the target sequences of primer set CPN 90/91 in the 16S rRNA gene of C. pneumoniae with the 16S rRNA genes of other respiratory pathogens. A dot indicates the same base, and a letter indicates a base different from that in C. pneumoniae. A dash indicates a deletion.

FIG. 3

Alignment of the target sequences of primer CPN 100 in the 16S rRNA gene of C. psittaci and primer CPN 101 in the 16S-23S spacer rRNA gene of C. psittaci with the 16S and 16S-23S spacer rRNA genes of other bacteria. A dot indicates the same base, and a letter indicates a base different from that in C. psittaci. A dash indicates a deletion.

FIG. 4

Analytical sensitivities of C. trachomatis TETR-PCR (primer set CTR 70/71) (top), C. pneumoniae TETR-PCR (primer set CPN 90/91) (middle), and C. psittaci TETR-PCR (primer set CPS 100/101) (bottom). The number of IFU is indicated at the top. Base-pair sizes of weight markers (wm) and PCR products are indicated in the margins. DNA from C. trachomatis VR 348 serovar E, C. pneumoniae AO3, and C. psittaci SM006 was amplified.

FIG. 5

Multiplex TETR-PCR products obtained for Chlamydia species with species-specific primer sets CTR 70/71, CPN 90/91, and CPS 100/101 targeting the 16S and 16S-23S spacer rRNA genes. One IFU per PCR was tested individually and in combinations. Base-pair sizes of weight markers (wm) and PCR products are indicated in the margins. DNA from C. trachomatis VR 348 serovar E, C. pneumoniae BAL 37, and C. psittaci SM006 was amplified.