Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions

Diagn Microbiol Infect Dis. 2003 Feb;45(2):85-95. doi: 10.1016/s0732-8893(02)00484-4.


Atypical pathogens such as Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae are an important cause of community-acquired pneumonia. The available detection methods (culture and serology) either lack sensitivity or give only a retrospective diagnosis. In order to improve their detection and quantification in respiratory samples, a real-time multiplex PCR, performed in two separate reactions, was developed for these three pathogens. The comparison of multiplex real-time and conventional PCR assay on 73 respiratory specimens showed an overall agreement of 98.3%, corresponding to 95.8%, 100% and 100% agreement for C. pneumoniae, L. pneumophila and M. pneumoniae, respectively. Clinical application of this multiplex real-time PCR was done on 40 respiratory samples from 38 patients with respiratory tract infections. Of 19 serology-positive patients, 14 were confirmed by the multiplex real-time PCR to be infected by either one of the three pathogens. All samples from serology-negative patients were negative with the multiplex real-time PCR.

MeSH terms

  • Biotechnology
  • Chlamydophila Infections / diagnosis
  • Chlamydophila pneumoniae / genetics
  • Chlamydophila pneumoniae / isolation & purification*
  • DNA, Bacterial / analysis
  • Humans
  • Legionella pneumophila / genetics
  • Legionella pneumophila / isolation & purification*
  • Legionnaires' Disease / diagnosis
  • Mycoplasma pneumoniae / genetics
  • Mycoplasma pneumoniae / isolation & purification*
  • Pneumonia, Bacterial / diagnosis*
  • Pneumonia, Mycoplasma / diagnosis
  • Polymerase Chain Reaction / methods*
  • Respiratory System / metabolism
  • Respiratory System / microbiology
  • Retrospective Studies
  • Sensitivity and Specificity


  • DNA, Bacterial