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Multiplex real-time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis.

ABSTRACT: BACKGROUND AND OBJECTIVE:Rapid diagnosis of pertussis is important for the timely isolation of the infection source and early prevention measures among the contact persons, especially among non-vaccinated infants for whom pertussis is life-threatening. MATERIALS AND METHODS:Targets IS481, IS1001, BP0026 and human GAPDH gene were used to develop a multiplex real-time PCR assay based on the TaqMan technology for detection and identification of Bordetella pertussis and Bordetella parapertussis in clinical samples. A total of 121 human clinical specimens obtained within 2012-2013 were used to evaluate the multiplex real-time PCR assay. Clinical specimens were also tested for culture and conventional PCR. Sensitivity and specificity for culture, conventional PCR, and multiplex real-time PCR were measured in comparison with a clinical standard for B. pertussis infection. RESULTS:The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for IS481, IS1001 and 10 genomic equivalents per reaction for BP0026 target. When the B. pertussis assays were compared with a clinical standard for B. pertussis infection, sensitivity was 5, 59 and 89% the specificity was 100, 100 and 100% for culture, conventional PCR, and multiplex real-time PCR, respectively. CONCLUSIONS:Developed multiplex real-time PCR offers a fast tool with high sensitivity and specificity for the diagnosis of B. pertussis and B. parapertussis infections which is suitable for implementation in a routine laboratory diagnostics.

SUBMITTER: Kolodkina V

PROVIDER: S-EPMC4393489 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Multiplex real-time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis.

Kolodkina Valentina V Martinov Vladimir V Babenko Andrey A

Iranian journal of microbiology 20140601 3

<h4>Background and objective</h4>Rapid diagnosis of pertussis is important for the timely isolation of the infection source and early prevention measures among the contact persons, especially among non-vaccinated infants for whom pertussis is life-threatening.<h4>Materials and methods</h4>Targets IS481, IS1001, BP0026 and human GAPDH gene were used to develop a multiplex real-time PCR assay based on the TaqMan technology for detection and identification of Bordetella pertussis and Bordetella par ...[more]

PMID: 25870746

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Project description:A rapid real-time multiplex PCR assay for detecting and differentiating Bordetella pertussis and Bordetella parapertussis in nasopharyngeal swabs was developed. This assay (LC-PCR-IS) targets the insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively, and is performed using the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.). The analytical sensitivity is less than one organism per reaction. Results for Bordetella culture and/or direct fluorescent antibody testing and a second LightCycler PCR assay (target, pertussis toxin gene) were compared to results of the LC-PCR-IS assay for 111 nasopharyngeal swabs submitted for pertussis testing. Of the specimens, 12 were positive (9 B. pertussis and 3 B. parapertussis) and 68 specimens were negative by all methods. Three other specimens were positive for B. pertussis by at least two of the methods (including the LC-PCR-IS assay), and another 28 specimens were positive for B. pertussis by the LC-PCR-IS assay only. No specimens were negative by the LC-PCR-IS assay and positive by the other methods. A conventional PCR method (target, IS481) was also compared to the LC-PCR-IS assay for a different group of nasopharyngeal swab specimens (n = 96): 44 specimens were positive and 41 specimens were negative for B. pertussis with both PCR methods. Nine specimens were positive for B. pertussis by the LC-PCR-IS assay and negative by the conventional PCR assay, and two specimens were positive for B. pertussis by the conventional PCR assay and negative by the LC-PCR-IS assay. Positivity of the two assays was not significantly different (P = 0.0654). The insertion sequence IS481 is also present in Bordetella holmesii; specimens containing B. holmesii may yield false-positive results. The LC-PCR-IS assay takes approximately 45 min to complete post-nucleic acid extraction, compared to 24 h for the conventional PCR assay previously used in our laboratory. The LC-PCR-IS assay is easier to perform than the conventional PCR assay, and the closed system decreases the chance of contamination. All of these characteristics represent a significant improvement in the detection of B. pertussis and B. parapertussis in nasopharyngeal specimens.

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

Multiplex real-time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis.

Publications

Multiplex real-time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis.

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