DETECTION OF CHLAMYDIA-PNEUMONIAE USING A GENERAL CHLAMYDIA POLYMERASE CHAIN-REACTION WITH SPECIES DIFFERENTIATION AFTER HYBRIDIZATION

A general polymerase chain reaction (PCR) for the detection of Chlamydia trachomatis, C. psittaci and C. pneumoniae was developed. By computer assisted sequence analysis two sets of sense primers CM1 and CM2 and one set of antisense primers CM3, each consisting of a mixture of two primers, were selected from conserved regions on the gene coding for the major outer membrane protein (MOMP-gene). Internal species specific oligoprobes were selected for the differentiation of the three species. The general PCR using primer combinations CMI/CM3 and CM2/CM3 generated a DNA fragment of approximately 360 and 320 bp respectively with 15 serovars of C. trachomatis, 6 strains of C. pneumoniae and 2 strains of C. psittaci. All amplified fragments hybridised with the general Chlamydia probe. The species specific probes detected only the Chlamydia strains of the corresponding species. Although both combinations gave rise to an identical specificity in the PCR, the CM2/CM3-PCR was 10-100 times more sensitive. The sensitivity of the CM2/CM3-PCR was one inclusion forming unit (IFU) or ten copies of isolated MOMP-DNA. Application of this general Chlamydia PCR on a throat sample weakly positive in culture for C. pneumoniae (one IFU) scored also positive after hybridisation with the general probe and the C. pneumoniae probe. The results indicate that the general Chlamydia PCR can be used for the sensitive detection of all Chlamydia species and could prove useful in the detection of Chlamydia pneumoniae in clinical specimens.