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Identification and evaluation of new target sequences for specific detection of Bordetella pertussis by real-time PCR.

ABSTRACT: A comparative analysis of the Bordetella pertussis, B. bronchiseptica, and B. parapertussis genome assemblies permitted the identification of regions with significant sequence divergence and the design of two new real-time PCR assays, BP283 and BP485, for the specific detection of B. pertussis. The performance characteristics of these two assays were evaluated and compared to those of culture and an existing real-time PCR assay targeting the repetitive element IS481. The testing of 324 nasopharyngeal specimens indicated that, compared to culture, the BP283 assay had a sensitivity and specificity of 100 and 96.8% and the BP485 assay had a sensitivity and specificity of 92.3 and 97.1%. Notably, B. holmesii was isolated from two specimens that were positive by the IS481 assay but negative by the BP283 and BP485 assays. These two assays represent an improvement in specificity over those of PCR assays targeting only IS481 and may be duplexed or used in conjunction with existing PCR assays to improve the molecular detection of B. pertussis.

SUBMITTER: Probert WS

PROVIDER: S-EPMC2566112 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Identification and evaluation of new target sequences for specific detection of Bordetella pertussis by real-time PCR.

Probert William S WS Ely Janet J Schrader Kimmi K Atwell Jessica J Nossoff Angela A Kwan Stanley S

Journal of clinical microbiology 20080827 10

A comparative analysis of the Bordetella pertussis, B. bronchiseptica, and B. parapertussis genome assemblies permitted the identification of regions with significant sequence divergence and the design of two new real-time PCR assays, BP283 and BP485, for the specific detection of B. pertussis. The performance characteristics of these two assays were evaluated and compared to those of culture and an existing real-time PCR assay targeting the repetitive element IS481. The testing of 324 nasophary ...[more]

PMID: 18753352

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Project description:BACKGROUND: Nucleic acid amplification of the IS481 region by PCR is more sensitive than culture for detection and diagnosis of Bordetella pertussis but the assay has known cross-reactivity for Bordetella holmesii and its use as a routine diagnostic assay has not been widely evaluated. METHODS: The objectives of this study were: 1) to assess the diagnostic utility of real-time IS481 PCR by comparison of results with culture and direct fluorescent antigen (DFA) testing for B. pertussis, 2) to employ a PCR assay designed against a different insertion sequence (IS1001) to assess the incidence of B. holmesii in symptomatic individuals and 3) to design and evaluate a new PCR-based assay which could be used for B. pertussis confirmation. A total of 808 nasopharyngeal specimens were included in the study the majority of which were submitted in charcoal transport medium (88%) with the rest submitted in Regan-Lowe medium. RESULTS: Concordant results for PCR, DFA and culture were obtained for 21 B. pertussis positive and 729 B. pertussis negative specimens. DFA was prone to false positive and negative reactions when compared with both PCR and culture. The IS481 PCR identified 28 positive results for specimens that were DFA and culture negative. A novel real-time PCR targeting the B. pertussis toxin promoter was found to be specific and useful for confirming the majority of IS481 positive specimens as B. pertussis. B. holmesii was not detected in any of the submitted samples. CONCLUSION: The potential pick up of B. holmesii by the IS481 PCR had minimal diagnostic relevance in the Alberta population during the time period of our study. The IS481 PCR assay is now used in our laboratory routinely for front-line screening of samples for B. pertussis with associated enhancement in diagnostic sensitivity compared with DFA and culture. Retrospectively, patients' samples are batched and tested by the IS1001 MB and TPR assays for research purposes and to ensure there is no change in B. holmesii incidence in the population.

Dataset Information

Identification and evaluation of new target sequences for specific detection of Bordetella pertussis by real-time PCR.

Publications

Identification and evaluation of new target sequences for specific detection of Bordetella pertussis by real-time PCR.

Similar Datasets

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