Evaluation of Amplification Targets for the Specific Detection of Bordetella pertussis Using Real-Time Polymerase Chain Reaction. Issue 4 (2014)

• Record Type: Journal Article
• Authors: Hasan, Mohammad Rubayet; Tan, Rusung; Al-Rawahi, Ghada N; Thomas, Eva; Tilley, Peter
• Is Part Of: Canadian journal of infectious diseases & medical microbiology =. Volume 25:Issue 4 (2014)
• Page Start: 217
• Page End: 221
• Publication Date: 2014
• Subjects: Bordetella pertussis -- IS481 element -- Pertactin gene -- Porin gene -- Ptx-promoter -- Real-time PCR; Communicable diseases -- Periodicals; Infection -- Periodicals; Communicable diseases; Infection; Communicable Diseases; Communicable Disease Control; Electronic journals; Periodicals; Fulltext; Internet Resources; Periodicals; 616.9
• DOI: 10.1155/2014/763128
• Languages: English
• ISSNs: 1712-9532

Abstract

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 IS 481, 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 IS 481, 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 IS 481 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. CONCLUSION: A novel porin assay for B pertussis demonstrated superior performance and may be useful for improved molecular detection of B pertussis in clinical specimens.