title: Evaluation of amplification targets for the specific detection of Bordetella pertussis using real-time polymerase chain reaction.
abstract: BACKGROUND
  Bordetella pertussis infections continue to be a major public health challenge in Canada. Polymerase chain reaction (PCR) assays to detect B pertussis are typically based on the multicopy insertion sequence IS481, which offers high sensitivity but lacks species specificity.
  
  
  METHODS
  A novel B pertussis real-time PCR assay based on the porin gene was tested in parallel with several previously published assays that target genes such as IS481, ptx-promoter, pertactin and a putative thialase. The assays were evaluated using a reference panel of common respiratory bacteria including different Bordetella species and 107 clinical nasopharyngeal specimens. Discrepant results were confirmed by sequencing the PCR products.
  
  
  RESULTS
  Analytical sensitivity was highest for the assay targeting the IS481 element; however, the assay lacked specificity for B pertussis in the reference panel and in the clinical samples. False-positive results were also observed with assays targeting the ptx-promoter and pertactin genes. A PCR assay based on the thialase gene was highly specific but failed to detect all reference strains of B pertussis. However, a novel assay targeting the porin gene demonstrated high specificity for B pertussis both in the reference panel and in clinical samples and, based on sequence-confirmed results, correctly predicted all B pertussis-positive cases in clinical samples. According to Probit regression analysis, the 95% detection limit of the new assay was 4 colony forming units/reaction.
  
  
  CONCLUSIONS
  A novel porin assay for B pertussis demonstrated superior performance and may be useful for improved molecular detection of B pertussis in clinical specimens.
authors.affiliation: Division of Microbiology, Virology and Infection Control, Department of Pathology, Children's and Women's Health Centre of BC; ; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia;
authors.forename: Mohammad Rubayet
authors.initials: MR
authors.lastname: Hasan
authors.affiliation: Division of Microbiology, Virology and Infection Control, Department of Pathology, Children's and Women's Health Centre of BC; ; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia; ; Sidra Medical and Research Center, Doha, Qatar.
authors.forename: Rusung
authors.initials: R
authors.lastname: Tan
authors.affiliation: Division of Microbiology, Virology and Infection Control, Department of Pathology, Children's and Women's Health Centre of BC; ; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia;
authors.forename: Ghada N
authors.initials: GN
authors.lastname: Al-Rawahi
authors.affiliation: Division of Microbiology, Virology and Infection Control, Department of Pathology, Children's and Women's Health Centre of BC; ; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia;
authors.forename: Eva
authors.initials: E
authors.lastname: Thomas
authors.affiliation: Division of Microbiology, Virology and Infection Control, Department of Pathology, Children's and Women's Health Centre of BC; ; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia;
authors.forename: Peter
authors.initials: P
authors.lastname: Tilley
keywords: Bordetella pertussis; IS481 element; Pertactin gene; Porin gene; Ptx-promoter; Real-time PCR