Comparison of sample preparation methods for detection of Chlamydia pneumoniae in bronchoalveolar lavage fluid by PCR.
Amplification inhibitors can lead to false-negative results for PCR. In order to evaluate the reliability of PCR for the detection of Chlamydia pneumoniae, the presence of PCR inhibitors in 75 bronchoalveolar lavage specimens was assessed after treatment by various sample preparation methods. Specimens were collected from patients with acute respiratory infections, including four cases of proven C. pneumoniae infection. Substances inhibitory to the amplification of chlamydial DNA continued to be present in 12% of the samples treated according to the commonly used single-step proteinase K digestion and in 31% of the samples processed by heat treatment. However, the complexing of DNA-contaminating proteins and polysaccharides from digested specimens to cetyltrimethylammonium bromide (CTAB) followed by DNA extraction efficiently removed inhibitors from all experimental samples and provided subsequent identification of all positive clinical samples by PCR. The CTAB method and proteinase K treatment had comparable detection limits of approximately 0.01 inclusion-forming units. CTAB-based DNA purification of respiratory specimens is recommended to increase the diagnostic sensitivity of PCR and confidence in negative results.
Maass
M
M
Institute of Medical Microbiology, Medical University of Lübeck, Germany.
Dalhoff
K
K
D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology*; D002460:Cell Line; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000494:pharmacology; D016993:Chlamydophila pneumoniae / Q000302:isolation & purification*; D019286:Endopeptidase K; D006358:Hot Temperature; D006801:Humans; D016133:Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000494:pharmacology; D013048:Specimen Handling
D002593:Cetrimonium Compounds; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D000077286:Cetrimonium