title: Direct PCR enables detection of Mycoplasma pneumoniae in patients with respiratory tract infections.
abstract: The sensitivities of three methods of detection of Mycoplasma pneumoniae by a 16S rDNA PCR were compared by using a serial dilution of M. pneumoniae. These methods consisted of a PCR performed directly on cells after a proteinase K pretreatment (direct PCR), a PCR after purification of nucleic acids (DNA-PCR), and a PCR with rRNA sequences as the target after reverse transcription. The direct PCR and the reverse transcription PCR had a sensitivity of 1.5 CFU (approximately 250 genomes). By purification, a 10-fold loss of target DNA occurred, as shown by a 10-fold decrease in sensitivity (15 CFU) of the DNA-PCR. The presence of an excess of human background DNA did not influence the sensitivity of either PCR. The direct PCR was evaluated on samples from patients with respiratory complaints. Direct PCR amplification was possible in 94.9% of the samples, which were tested by amplification of a 326-bp fragment of the beta-globin gene, which was performed to test for the suitability of amplification. Nucleic acid purification was performed on the beta-globin-negative samples, after which only 2% remained negative. A positive correlation between the direct M. pneumoniae PCR and serology, as tested by the microparticle agglutination assay (MAG assay), was found in 88.1% of the cases. A positive MAG assay result was found for samples from 10 (17%) of the patients; samples from 6 (10.2%) of these patients were also positive by PCR. Samples from three patients were found to be positive by the M. pneumoniae PCR and negative by the MAG assay. Persistence of M. pneumoniae, as detected by PCR was observed in three patients. These results indicate that the direct PCR with 16S rDNA could prove to be useful in the detection of M. pneumoniae in respiratory tract samples, although more studies are needed to evaluate the correlation between clinical symptoms and positive test results.
authors.affiliation: Department of Clinical Microbiology, Free University Hospital, Amsterdam, The Netherlands.
authors.forename: J H
authors.initials: JH
authors.lastname: Tjhie
authors.forename: F J
authors.initials: FJ
authors.lastname: van Kuppeveld
authors.forename: R
authors.initials: R
authors.lastname: Roosendaal
authors.forename: W J
authors.initials: WJ
authors.lastname: Melchers
authors.forename: R
authors.initials: R
authors.lastname: Gordijn
authors.forename: D M
authors.initials: DM
authors.lastname: MacLaren
authors.forename: J M
authors.initials: JM
authors.lastname: Walboomers
authors.forename: C J
authors.initials: CJ
authors.lastname: Meijer
authors.forename: A J
authors.initials: AJ
authors.lastname: van den Brule
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000372:Agglutination Tests; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D004275:DNA, Ribosomal / Q000235:genetics; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008969:Molecular Sequence Data; D009177:Mycoplasma pneumoniae / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012194:RNA-Directed DNA Polymerase; D012141:Respiratory Tract Infections / Q000382:microbiology*; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
chemical_list: D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012194:RNA-Directed DNA Polymerase