title: 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.
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 chronic cough. We found the same detection limit for the two methods and 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 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 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 this 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 work 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 chronic cough.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Aarhus, Denmark. mygind@biobase.dk
authors.forename: Tina
authors.initials: T
authors.lastname: Mygind
authors.forename: Svend
authors.initials: S
authors.lastname: Birkelund
authors.forename: Niels H
authors.initials: NH
authors.lastname: Birkebaek
authors.forename: Lars
authors.initials: L
authors.lastname: Østergaard
authors.forename: Jørgen Skov
authors.initials: JS
authors.lastname: Jensen
authors.forename: Gunna
authors.initials: G
authors.lastname: Christiansen
mesh_terms: D002411:Cation Exchange Resins / Q000378:metabolism; D002614:Chelating Agents / Q000378:metabolism*; D016993:Chlamydophila pneumoniae / Q000235:genetics* / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D006367:HeLa Cells / Q000821:virology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D012117:Resins, Synthetic
chemical_list: D002411:Cation Exchange Resins; D002614:Chelating Agents; D004269:DNA, Bacterial; D012117:Resins, Synthetic; C024997:Chelex 100