title: A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa.
abstract: BACKGROUND
  Chronic lung infection with the bacterium Pseudomonas aeruginosa is one of the hallmarks of cystic fibrosis (CF) and is associated with worsening lung function, increased hospitalisation and reduced life expectancy. A virulent clonal strain of P. aeruginosa (Australian epidemic strain I; AES-I) has been found to be widespread in CF patients in eastern Australia.
  
  
  METHODS
  Suppression subtractive hybridization (SSH) was employed to identify genetic sequences that are present in the AES-I strain but absent from the sequenced reference strain PAO1. We used PCR to evaluate the distribution of several of the AES-I loci amongst a collection of 188 P. aeruginosa isolates which was comprised of 35 AES-I isolates (as determined by PFGE), 78 non-AES-I CF isolates including other epidemic CF strains as well as 69 P. aeruginosa isolates from other clinical and environmental sources.
  
  
  RESULTS
  We have identified a unique AES-I genetic locus that is present in all 35 AES-I isolates tested and not present in any of the other 153 P. aeruginosa strains examined. We have used this unique AES-I locus to develop a diagnostic PCR and a real-time PCR assay to detect the presence of P. aeruginosa and AES-I in patient sputum samples.
  
  
  CONCLUSIONS
  We have developed diagnostic PCR assays that are 100% sensitive and 100% specific for the P. aeruginosa strain AES-I. We have also shown that Whatman FTA Elute cards may be used with PCR-based assays to rapidly detect the presence of P. aeruginosa strains in CF sputum.
authors.affiliation: Department of Microbiology, Monash University, VIC 3800, Australia.
authors.forename: Heidi L
authors.initials: HL
authors.lastname: Williams
authors.forename: Lynne
authors.initials: L
authors.lastname: Turnbull
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Thomas
authors.forename: Anna
authors.initials: A
authors.lastname: Murphy
authors.forename: Tim
authors.initials: T
authors.lastname: Stinear
authors.forename: David S
authors.initials: DS
authors.lastname: Armstrong
authors.forename: Cynthia B
authors.initials: CB
authors.lastname: Whitchurch
mesh_terms: D001315:Australia / Q000453:epidemiology; D001431:Bacteriological Techniques / Q000379:methods*; D002648:Child; D003550:Cystic Fibrosis / Q000150:complications*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D006801:Humans; D009693:Nucleic Acid Hybridization; D018410:Pneumonia, Bacterial / Q000453:epidemiology* / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011552:Pseudomonas Infections / Q000453:epidemiology* / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial