Comparison of five PCR assays for detecting Chlamydophila pneumoniae DNA.
We compared five different polymerase chain reaction (PCR) assays for the detection of Chlamydophila pneumoniae DNA using highly purified elementary bodies (EBs) and peripheral blood mononuclear cells (PBMCs) from healthy blood donors. The primers were as follows; two targeting the 16S rRNA gene, one targeting the ompA gene, one targeting the Pst-I gene, and one targeting the 53 kDa outer membrane protein gene. The 16S rRNA touchdown enzyme time release (TETR) PCR, the ompA nested PCR and the 53 kDa nested PCR were the most sensitive assays and could detect one or more EB per assay. These three PCRs also had the same reproducibility, but the minimal amount of C. pneumoniae that could be reproducibly detected (10 of 10 testing positive) was 20 EBs. In a sample of specimens from healthy blood donors, we found 5 of 77 (6.5%) PBMCs specimens to have C. pneumoniae DNA according to the nested ompA PCR. Specimens with the 16S rRNA TETR and 53 kDa nested assays were found to have C. pneumoniae DNA 7 of 77 (9.1%) and 18 of 77 (23.4%) specimens, respectively. The other two assays failed to detect even a single positive. However, the detection rate decreased with repeated testing of the same samples. Our newly designed 53 kDa nested PCR may be as useful as the other four recommended PCR assays and may be a more useful assay for the detection of C. pneumoniae DNA from PBMCs.
Fukano
Hiroshi
H
Division of Respiratory Diseases, Department of Medicine, Kawasaki Medical School, Kurashiki, Okayama, Japan. fukano@med.kawasaki-m.ac.jp
D000328:Adult; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000235:genetics; D001782:Blood Donors; D016993:Chlamydophila pneumoniae / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis* / Q000097:blood; D015252:Deoxyribonucleases, Type II Site-Specific; D005260:Female; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000382:microbiology; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000235:genetics; D015203:Reproducibility of Results
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; C078167:OMPA outer membrane proteins; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific