title: Multiplex PCR for the simultaneous detection of Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila in community-acquired pneumonia.
abstract: A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection of Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila. Oligonucleotide primers for the amplification of the DNA of these three organisms were optimized for use in combination in the same reaction. PCR products were detected by the Micro-Chip Electrophoresis Analysis System. Clinical samples were obtained from 208 community-acquired pneumonia (CAP) patients who were participants in a multicenter CAP surveillance study performed at seven medical schools and their affiliate hospitals in Japan. No significant differences in the sensitivity of each primer set were observed when tested in both the multiplex and monoplex PCR assays. Our multiplex PCR was able to reliably detect 10 copies/100 microl of each of the three pathogen DNAs. Of the panel of 208 samples, 14 of 15 C. pneumoniae, 10 of 10 M. pneumoniae, eight of eight L. pneumophila and 165 of 176 negative samples were correctly identified. Eleven cases who were the multiplex PCR positive and conventional method negative were observed. The PCR findings were of possible significance in at least four of these patients. Our multiplex PCR assay could potentially be used as a diagnostic and epidemiological tool. Further prospective studies are needed to establish its clinical usefulness.
authors.affiliation: Division of Respiratory Diseases, Department of Internal Medicine, Kawasaki Medical School, 577 Matushima, Kurashiki City, Okayama 701-0192, Japan. nao@med.kawasaki-m.ac.jp
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Miyashita
authors.forename: Atsushi
authors.initials: A
authors.lastname: Saito
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kohno
authors.forename: Keizo
authors.initials: K
authors.lastname: Yamaguchi
authors.forename: Akira
authors.initials: A
authors.lastname: Watanabe
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Oda
authors.forename: Yukumasa
authors.initials: Y
authors.lastname: Kazuyama
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Matsushima
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D023521:Chlamydophila Infections / Q000175:diagnosis*; D017714:Community-Acquired Infections / Q000175:diagnosis; D005260:Female; D006801:Humans; D016952:Legionella pneumophila; D007877:Legionnaires' Disease / Q000175:diagnosis*; D008297:Male; D008875:Middle Aged; D009177:Mycoplasma pneumoniae; D018410:Pneumonia, Bacterial / Q000175:diagnosis*; D011019:Pneumonia, Mycoplasma / Q000175:diagnosis; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity