title: Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reaction assay targeting the ecfX and the gyrB genes.
abstract: Phenotypic identification of Gram-negative bacteria from respiratory specimens of patients with cystic fibrosis carries a high risk of misidentification. Molecular identification techniques that use single-gene targets are also susceptible to error, including cross-reaction issues with other Gram-negative organisms. In this study, we have designed a Pseudomonas aeruginosa duplex real-time polymerase chain reaction (PCR) (PAduplex) assay targeting the ecfX and the gyrB genes. The PAduplex was evaluated against a panel of 91 clinical and environmental isolates that were presumptively identified as P. aeruginosa. The results were compared with those obtained using a commercial biochemical identification kit and several other P. aeruginosa PCR assays. The results showed that the PAduplex assay is highly suitable for routine identification of P. aeruginosa isolates from clinical or environmental samples. The 2-target format provides simultaneous confirmation of P. aeruginosa identity where both the ecfX and gyrB PCR reactions are positive and may also reduce the potential for false negatives caused by sequence variation in primer or probe targets.
authors.affiliation: Department of Paediatrics and Child Health, University of Queensland, Brisbane 4029, Australia.
authors.forename: Snehal N
authors.initials: SN
authors.lastname: Anuj
authors.forename: David M
authors.initials: DM
authors.lastname: Whiley
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Kidd
authors.forename: Scott C
authors.initials: SC
authors.lastname: Bell
authors.forename: Claire E
authors.initials: CE
authors.lastname: Wainwright
authors.forename: Michael D
authors.initials: MD
authors.lastname: Nissen
authors.forename: Theo P
authors.initials: TP
authors.lastname: Sloots
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D015373:Bacterial Typing Techniques / Q000379:methods; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D003550:Cystic Fibrosis / Q000150:complications / Q000382:microbiology; D027081:DNA Gyrase / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction; D011552:Pseudomonas Infections / Q000150:complications / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000302:isolation & purification*; D012808:Sigma Factor / Q000235:genetics*; D013183:Sputum / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012808:Sigma Factor; D027081:DNA Gyrase