title: Development of a diagnostic PCR assay that targets a heat-shock protein gene (groES) for detection of Pseudomonas spp. in cystic fibrosis patients.
abstract: Laboratory detection of Pseudomonas spp., in particular Pseudomonas aeruginosa, remains an important assay in the management of patients with cystic fibrosis (CF). As the groES and groEL genes of P. aeruginosa have now been cloned and their nucleotide sequences determined, the aim of this study was to develop a novel PCR assay for the detection of Pseudomonas spp. from patients with CF by employing conserved primer regions of the groE heat-shock protein domain gene. A PCR assay was designed that targeted a 536 bp region of the groE gene to detect Pseudomonas spp. PCR amplification of genomic DNA from extracted organisms generated an amplicon of the expected size (approx. 536 bp) for all P. aeruginosa (n = 60), Pseudomonas putida, Pseudomonas fluorescens and Pseudomonas stutzeri isolates examined, but did not produce a positive amplicon for several other genera and species that are commonly isolated from the sputum of CF patients. RFLP analysis of the amplicons of all P. aeruginosa isolates demonstrated a single RFLP type that consisted of three bands at approximately 80, 190 and 250 bp; direct sequencing of the amplicons demonstrated the presence of a single sequence type, indicating the highly conserved nature of this region. In addition, the assay successfully produced a positive signal from primary non-selective plates of three known P. aeruginosa culture-positive CF patients, but was unable to generate a signal in a further six CF patients who had no history of infection with P. aeruginosa or other Pseudomonas spp. This assay is recommended to detect the presence of Pseudomonas spp., including P. aeruginosa, from primary culture plates that originate from laboratory analysis of CF patients' sputum, particularly at review, in those patients with no previous history of Pseudomonas infection or those who appear to be transiently colonized by this organism. Employment of such molecular methodologies, in conjunction with routine clinical sputum cultures, may provide improved information on the microbial status of CF patients, which will aid clinicians in both optimum patient management in terms of antibiotic regimes and CF centre infection-control practices.
authors.affiliation: Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, UK 2Northern Ireland Regional Adult Cystic Fibrosis Centre, Belfast City Hospital, Belfast BT9 7AB, Northern Ireland, UK 3Department of Respiratory Medicine, Prince of Wales Hospital, Brisbane, Queensland, Australia 4Infectious Diseases Laboratory, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5Department of Respiratory Medicine, The Queen's University of Belfast, Level 8, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, Northern Ireland, UK.
authors.forename: Lena
authors.initials: L
authors.lastname: Clarke
authors.affiliation: Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, UK 2Northern Ireland Regional Adult Cystic Fibrosis Centre, Belfast City Hospital, Belfast BT9 7AB, Northern Ireland, UK 3Department of Respiratory Medicine, Prince of Wales Hospital, Brisbane, Queensland, Australia 4Infectious Diseases Laboratory, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5Department of Respiratory Medicine, The Queen's University of Belfast, Level 8, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, Northern Ireland, UK.
authors.forename: John E
authors.initials: JE
authors.lastname: Moore
authors.affiliation: Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, UK 2Northern Ireland Regional Adult Cystic Fibrosis Centre, Belfast City Hospital, Belfast BT9 7AB, Northern Ireland, UK 3Department of Respiratory Medicine, Prince of Wales Hospital, Brisbane, Queensland, Australia 4Infectious Diseases Laboratory, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5Department of Respiratory Medicine, The Queen's University of Belfast, Level 8, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, Northern Ireland, UK.
authors.forename: B Cherie
authors.initials: BC
authors.lastname: Millar
authors.affiliation: Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, UK 2Northern Ireland Regional Adult Cystic Fibrosis Centre, Belfast City Hospital, Belfast BT9 7AB, Northern Ireland, UK 3Department of Respiratory Medicine, Prince of Wales Hospital, Brisbane, Queensland, Australia 4Infectious Diseases Laboratory, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5Department of Respiratory Medicine, The Queen's University of Belfast, Level 8, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, Northern Ireland, UK.
authors.forename: Luke
authors.initials: L
authors.lastname: Garske
authors.affiliation: Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, UK 2Northern Ireland Regional Adult Cystic Fibrosis Centre, Belfast City Hospital, Belfast BT9 7AB, Northern Ireland, UK 3Department of Respiratory Medicine, Prince of Wales Hospital, Brisbane, Queensland, Australia 4Infectious Diseases Laboratory, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5Department of Respiratory Medicine, The Queen's University of Belfast, Level 8, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, Northern Ireland, UK.
authors.forename: Jiru
authors.initials: J
authors.lastname: Xu
authors.affiliation: Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, UK 2Northern Ireland Regional Adult Cystic Fibrosis Centre, Belfast City Hospital, Belfast BT9 7AB, Northern Ireland, UK 3Department of Respiratory Medicine, Prince of Wales Hospital, Brisbane, Queensland, Australia 4Infectious Diseases Laboratory, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5Department of Respiratory Medicine, The Queen's University of Belfast, Level 8, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, Northern Ireland, UK.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Heuzenroeder
authors.affiliation: Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, UK 2Northern Ireland Regional Adult Cystic Fibrosis Centre, Belfast City Hospital, Belfast BT9 7AB, Northern Ireland, UK 3Department of Respiratory Medicine, Prince of Wales Hospital, Brisbane, Queensland, Australia 4Infectious Diseases Laboratory, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5Department of Respiratory Medicine, The Queen's University of Belfast, Level 8, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, Northern Ireland, UK.
authors.forename: Mary
authors.initials: M
authors.lastname: Crowe
authors.affiliation: Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, UK 2Northern Ireland Regional Adult Cystic Fibrosis Centre, Belfast City Hospital, Belfast BT9 7AB, Northern Ireland, UK 3Department of Respiratory Medicine, Prince of Wales Hospital, Brisbane, Queensland, Australia 4Infectious Diseases Laboratory, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5Department of Respiratory Medicine, The Queen's University of Belfast, Level 8, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, Northern Ireland, UK.
authors.forename: J Stuart
authors.initials: JS
authors.lastname: Elborn
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D018835:Chaperonin 10 / Q000737:chemistry / Q000235:genetics*; D003470:Culture Media; D003550:Cystic Fibrosis / Q000382:microbiology*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D011549:Pseudomonas / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D011552:Pseudomonas Infections / Q000175:diagnosis* / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013183:Sputum / Q000382:microbiology
chemical_list: D018835:Chaperonin 10; D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial