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Open Access Highly Accessed Research article

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

Tina Mygind1*, Svend Birkelund1, Niels H Birkebæk2, Lars Østergaard3, Jørgen Skov Jensen4 and Gunna Christiansen1

Author Affiliations

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

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BMC Microbiology 2002, 2:17  doi:10.1186/1471-2180-2-17

Published: 9 July 2002

Abstract

Background

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.

Results

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.

Conclusions

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.