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Title: ANALYTICAL VERIFICATION OF A PCR FOR IDENTIFICATION OF BORDETELLA AVIUM

Author
item Register, Karen
item Yersin, Andrew

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/15/2005
Publication Date: 11/15/2005
Citation: Register, K.B., Yersin, A.G. 2005. Analytical verification of a PCR for identification of Bordetella avium. Journal of Clinical Microbiology. 43(11):5567-5573.

Interpretive Summary: The bacterium Bordetella avium causes turkey coryza or bordetellosis, a respiratory disease responsible for substantial economic losses to the turkey industry. The methods currently used to identify B. avium are not optimal because of the lengthy time required and the subjective nature of some steps. Identification methods based on PCR are being increasingly used by clinical laboratories since they are often more sensitive and specific and can be accomplished in a much shorter period of time than traditional methods. This report describes the optimal conditions and performance characteristics of a PCR for identification of B. avium. This assay is highly sensitive and specific and can be accomplished in a few hours, as compared to several days for currently used methods. Rapid detection of B. avium will be of value in investigating and controlling outbreaks.

Technical Abstract: Background: Bordetella avium is the etiologic agent of 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. A PCR for detection of B. avium was proposed a number of years previously (Savelkoul, P. H. M., L. E. G. M. de Groot, C. Boersma, I. Livey, C. J. Duggleby, B. A. M. van der Zeijst, and W. Gaastra. 1993. Microb. Pathog. 15:207-215), but absence of detail and lack of analytical verification preclude its use as a diagnostic tool. In the present study we have identified an optimal set of reaction conditions for use with the previously described primer pair and determined the performance characteristics of the assay. Methods: 72 B. avium isolates, from diverse geographic locations and covering a time span of at least 25 years, and 85 poultry isolates of other genera and species, representing both pathogens and commensals, were evaluated. PCR optimization and analysis of performance characteristics were carried out in an ABI 9700 thermal cycler. Boiled cell lysates or genomic DNA was used as template. PCR results were compared with the gold standard of culture and biochemical testing. PCR products were directly sequenced by fluorescence-based cycle sequencing with AmpliTaq and BigDye Terminators on an ABI 377 sequencer. Results: Using the optimized conditions identified, assay sensitivity is 100% and specificity is 98.6%. Reproducibility is 100% with genomic DNA or with cell lysates less than 3 days old. The limit of detection is <20pg. Sequence analysis of the B. avium amplicons identified 3 variants, which share 99.5% identity. Conclusion: These data indicate the B. avium PCR assay can be expected to provide clinically significant results, thereby enhancing biosecurity measures through more rapid detection of outbreaks.