Dataset Information

Analytical verification of a PCR assay for identification of Bordetella avium.

ABSTRACT: Bordetella avium is the etiologic agent of turkey coryza or bordetellosis, a respiratory disease responsible for substantial economic losses to the turkey industry. At present, identification of this bacterium relies on isolation and biochemical testing. Although a PCR for the detection of B. avium was proposed a number of years ago, lack of analytical verification precludes its use as a diagnostic tool. Furthermore, a number of details pertaining to the reaction conditions used are missing or unclear. In the present study we have identified an optimal set of PCR conditions for use with the previously described primer pair and determined the limit of detection under these conditions to be approximately 20 pg. Assay sensitivity is 100%, based on an analysis of 72 B. avium isolates from diverse geographic locations and covering a time span of at least 25 years. Evaluation of a separate group of 87 bacterial isolates from poultry, comprising both gram-positive and gram-negative commensals and pathogens representing 11 genera, demonstrated an assay specificity of 98.8%. Reproducibility is 100% using either purified genomic DNA or boiled cell lysates less than 3 days old. Sequence analysis of the B. avium PCR amplicons identified only three occasional sequence polymorphisms. These data indicate the B. avium PCR assay can provide clinically significant results.

SUBMITTER: Register KB

PROVIDER: S-EPMC1287781 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Analytical verification of a PCR assay for identification of Bordetella avium.

Journal of clinical microbiology 20051101 11

Bordetella avium is the etiologic agent of turkey coryza or bordetellosis, a respiratory disease responsible for substantial economic losses to the turkey industry. At present, identification of this bacterium relies on isolation and biochemical testing. Although a PCR for the detection of B. avium was proposed a number of years ago, lack of analytical verification precludes its use as a diagnostic tool. Furthermore, a number of details pertaining to the reaction conditions used are missing or u ...[more]

PMID: 16272488

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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.

Dataset Information

Analytical verification of a PCR assay for identification of Bordetella avium.

Publications

Analytical verification of a PCR assay for identification of Bordetella avium.

Similar Datasets

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