Development of a simplified polymerase chain reaction-enzyme immunoassay for the detection of Chlamydia pneumoniae

The 16S rRNA genes of two Chlamydia pneumoniae and two C. psittaci strains of different serovars were sequenced then compared to previously reported Chlamydia 16S rRNA gene sequences. Chlamydia pneumoniae-specific regions were identified and specific primers for nested PCR were synthesized. Nested PCR reactions were performed, in a single tube, by varying the annealing temperature of the amplification cycles. The initial thermal cycles were selected to allow annealing and extension of only the outer primer pair, whilst in later cycles a temperature that allowed inner primer annealing was employed. The inner primers were labelled, one with biotin and the other with fluorescein and consequently the dual labelled amplicon could be immobilized onto antibiotin-coated microtitre plates and detected colorimetrically via an antifluorescein-enzyme conjugate. The assay was found to be sensitive and specific. No cross reactions were observed with C. trachomatis, C. psittaci or other common respiratory pathogens.