Determination of PCR efficiency in chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae
1 Department of Medical Microbiology and Immunology, The Bartholin Building, University of Aarhus, Wilhelm Meyers Alle, DK-8000 Aarhus C, Denmark
2 Department of Pediactrics, Skejby Hospital, University Hospital of Aarhus, Brendstrupgaardsvej, DK-8200 Aarhus N, Denmark
3 Research Unit Q of Infectious Diseases, Skejby Hospital, University Hospital of Aarhus, Brendstrupgaardsvej, DK-8200 Aarhus N, Denmark
4 Department of Respiratory Infections, Meningitis and STI, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark
BMC Microbiology 2002, 2:17 doi:10.1186/1471-2180-2-17Published: 9 July 2002
Chlamydia pneumoniae infection has been detected by serological methods, but PCR is gaining more interest. A number of different PCR assays have been developed and some are used in combination with serology for diagnosis. Real-time PCR could be an attractive new PCR method; therefore it must be evaluated and compared to conventional PCR methods.
We compared the performance of a newly developed real-time PCR with a conventional PCR method for detection of C. pneumoniae. The PCR methods were tested on reference samples containing C. pneumoniae DNA and on 136 nasopharyngeal samples from patients with a chronic cough. We found the same detection limit for the two methods and that clinical performance was equal for the real-time PCR and for the conventional PCR method, although only three samples tested positive. To investigate whether the low prevalence of C. pneumoniae among patients with a chronic cough was caused by suboptimal PCR efficiency in the samples, PCR efficiency was determined based on the real-time PCR. Seventeen of twenty randomly selected clinical samples had a similar PCR efficiency to samples containing pure genomic C. pneumoniae DNA.
These results indicate that the performance of real-time PCR is comparable to that of conventional PCR, but that needs to be confirmed further. Real-time PCR can be used to investigate the PCR efficiency which gives a rough estimate of how well the real-time PCR assay works in a specific sample type. Suboptimal PCR efficiency of PCR is not a likely explanation for the low positivity rate of C. pneumoniae in patients with a chronic cough.