title: Real-time quantitative PCR for the detection of Streptococcus pneumoniae in the middle ear fluid of children with acute otitis media.
abstract: PCR based on the amplification of pneumolysin gene fragments has previously been applied to demonstrate Streptococcus pneumoniae in clinical specimens. Here, a real-time PCR method for the detection and quantification of pneumococci by amplifying a 206-bp fragment of the pneumolysin-encoding gene is described. The amplified fragments were detected simultaneously using fluorescent-labeled sequence-specific hybridization probes. The applicability of the assay to clinical samples was evaluated by studying 50 middle ear fluid (MEF) specimens from children with acute otitis media. Twenty-six of the MEF samples were positive by real-time PCR and the numbers of genome equivalents detected varied from 90 to 88,000/microl in 17 culture-positive samples and from 1 to 1,200/microl in 9 culture-negative samples. The results were compared to culture findings and to results obtained by using agarose gel electrophoresis or Europium-labeled hybridization probes for the detection of amplification products of conventional PCR. The sensitivity and specificity of the real-time PCR assay developed in the present study compared to culture were 100 and 73%, and to conventional PCR with agarose gel and/or TRF detection 93 and 96%, respectively. The real-time PCR assay was found to be rapid, easy to use, and sensitive in detecting and quantifying pneumococci.
authors.affiliation: National Public Health Institute (KTL), Oulu, Finland. Annika.Saukkoriipi@ktl.fi
authors.forename: Annika
authors.initials: A
authors.lastname: Saukkoriipi
authors.forename: Arto
authors.initials: A
authors.lastname: Palmu
authors.forename: Terhi
authors.initials: T
authors.lastname: Kilpi
authors.forename: Maija
authors.initials: M
authors.lastname: Leinonen
mesh_terms: D000208:Acute Disease; D001426:Bacterial Proteins; D002571:Cerumen / Q000382:microbiology*; D002648:Child; D004269:DNA, Bacterial / Q000032:analysis; D005063:Europium; D005456:Fluorescent Dyes; D006801:Humans; D010034:Otitis Media with Effusion / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000302:isolation & purification*; D013301:Streptolysins / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae; D005063:Europium