Polymerase chain reaction amplification of major outer membrane protein and 16S rRNA genes for detection of Chlamydia pneumoniae

Abstract

Chlamydia pneumoniae is a common cause of respiratory tract infection and community-acquired pneumonia. Diagnosis from clinical presentation cannot differentiate from infection caused by other bacteria or viruses. The rapid laboratory method for diagnosis of c. pneumoniae infection is important for effective treatment and prevention of chronic infection. To assess the utility of polymerase chain reaction (PCR) in detection of c. pneumoniae DNA from clinical specimens for diagnosis of acute infection with c. pneumoniae, two sets of primers were used for the direct species-specific detection of the major outer membrane protein (omp1) and 16S rRNA genes of c. pneumoniae. Two PCR protocols were employed; nested PCR and single-step PCR accompanied by dot blot hybridization. The false positive result due to amplicon carryover was prevented by the enzymatic degradation procedure with the use of uracil-Nglycosylase (UNG) in the first amplification. The recombinant plasmid controls were constructed and used for the detection of inhibitors in the samples. The sensitivity in the detection of purified c. pneumoniae DNA for omp1-based PCR and 16S rDNA-based PCR was 1 IFU and 0.1 IFU, respectively. We compared omp1-based PCR and 16S rDNA based PCR with indirect fluorescent-antibody (IFA) stain, and serology by Microimmunofluorescence (MIF) test. These tests were applied to 115 throat swab specimens and sera collected from 114 patients with community-acquired pneumonia. Fifteen specimens (13.0 %) from 14 patients (12.3 %) gave the positive results. None of the throat swabs samples was c. pneumoniae positive by IFA stain. Twelve throat swab samples that PCR positive obtained from 11 patients who had positive results in MIF test. Three specimens had PCR positive results without serologic evidence of acute infection and were considered to be false positive. When using 16S rDNA nested PCR, we could detect c. pneumoniae DNA from all of 15 positive samples. Of these 15 throat swab samples, 10 were positive by ompl-based PCR when analyzed with dot blot hybridization and all of them were negative by omp1 nested PCR. PCR inhibitors were detected in 3.5% of all samples and after dilution, all of them gave negative result. This data indicates that 16S rDNA-nested PCR can be established as a rapid and reliable method for the diagnosis of acute infection with c. pneumoniae.