title: Development of conventional and real-time nucleic acid sequence-based amplification assays for detection of Chlamydophila pneumoniae in respiratory specimens.
abstract: Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Chlamydophila pneumoniae 16S rRNA by using the NucliSens basic kit (bioMérieux, Boxtel, The Netherlands). The assay was originally developed as a conventional NASBA assay with electrochemiluminescence detection and was subsequently adapted to a real-time NASBA format by using a molecular beacon. C. pneumoniae RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild-type C. pneumoniae RNA and 0.1 inclusion-forming unit (IFU) of C. pneumoniae. In spiked respiratory specimens, the sensitivity of the C. pneumoniae NASBA assay varied between 0.1 and 1 IFU/100 mul sample, depending on the type of specimen. Finally, conventional and real-time NASBA were applied to respiratory specimens previously tested by PCR. A 100% concordance between the test results was obtained.
authors.affiliation: Department of Medical Microbiology, University of Antwerp, Universiteitsplein 1 S3, B-2610 Wilrijk, Belgium. katherine.loens@ua.ac.be
authors.forename: K
authors.initials: K
authors.lastname: Loens
authors.forename: T
authors.initials: T
authors.lastname: Beck
authors.forename: H
authors.initials: H
authors.lastname: Goossens
authors.forename: D
authors.initials: D
authors.lastname: Ursi
authors.forename: M
authors.initials: M
authors.lastname: Overdijk
authors.forename: P
authors.initials: P
authors.lastname: Sillekens
authors.forename: M
authors.initials: M
authors.lastname: Ieven
mesh_terms: D016993:Chlamydophila pneumoniae / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D012137:Respiratory System / Q000382:microbiology*; D021161:Self-Sustained Sequence Replication; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S