A new DIG-PCR-EIA method for the detection of Chlamydia pneumoniae DNA in clinical samples.
Chlamydia pneumoniae, a common respiratory pathogen, may also play a role in the pathogenesis of other chronic conditions. For accurate detection of infected persons and verification of results obtained by other PCR methods, a DIG-PCR-EIA method was evaluated. In the DIG-PCR-EIA, a 437 bp DNA sequence was amplified and hybridized with a newly synthesized 229 bp biotin-labeled probe. The end product was detected by an enzyme immunoassay. The sensitivity of DIG-PCR-EIA was compared with Southern blot hybridization and one-step HR/HL PCR, which was the routine method used. DNA was detected to the level of 20 elementary bodies of DIG-EIA-PCR compared to less than 2 by Southern blot, and 200 by HR/HL PCR. Thus a 100-fold increase in sensitivity could be expected by DIG-EIA-PCR compared to the routine method. Throat swabs and adenoid tissue from 22 children with otitis and middle ear secretions from 29 children, as well as throat swabs from 179 blood donors, were analyzed with DIG-EIA-PCR, HL/HR PCR and nested touchdown PCR. 32% of the ear secretions were positive by DIG-EIA-PCR as compared to 5% by the other two methods. Three adenoid tissue samples were positive by all methods applied. Among the child and adult throat samples, 18% and 32%, respectively, were positive by DIG-EIA-PCR and 5% and 10% by HR/HLPCR. The results indicate the suitability of DIG-PCR-EIA for verification of results of HR/HL PCR. DIG-PCR-EIA has a potential for increased sensitivity and adaptation for automation. It should be further evaluated using various types of tissue specimens and DNA extraction methods.
Gnarpe
J
J
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Canada. judy.gnarpe@ualberta.ca
Lindquist
L
L
D000234:Adenoids / Q000382:microbiology; D000328:Adult; D019921:Biotinylation; D015139:Blotting, Southern; D002353:Carrier State / Q000382:microbiology; D002648:Child; D002690:Chlamydia Infections / Q000382:microbiology*; D016993:Chlamydophila pneumoniae / Q000235:genetics / Q000302:isolation & purification*; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis*; D004076:Digoxigenin / Q000032:analysis*; D004432:Ear, Middle / Q000382:microbiology; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D010033:Otitis Media / Q000382:microbiology; D010614:Pharynx / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012141:Respiratory Tract Infections / Q000382:microbiology; D012680:Sensitivity and Specificity; D019809:Streptavidin
D015342:DNA Probes; D004269:DNA, Bacterial; D019809:Streptavidin; D004076:Digoxigenin