Real-time quantitative PCR for the detection of Streptococcus pneumoniae in the middle ear fluid of children with acute otitis media

被引:44
作者
Saukkoriipi, A
Palmu, A
Kilpi, T
Leinonen, M
机构
[1] Natl Publ Hlth Inst KTL, FIN-90101 Oulu, Finland
[2] Natl Publ Hlth Inst KTL, Helsinki, Finland
关键词
real-time PCR; Streptococcus pneumoniae;
D O I
10.1006/mcpr.2002.0443
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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/mul in 17 culture-positive samples and from 1 to 1200/mul 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. (C) 2002 Elsevier Science Ltd. All rights reserved.
引用
收藏
页码:385 / 390
页数:6
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