Analytical verification of a PCR assay for identification of Bordetella avium.
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.
Register
Karen B
KB
Respiratory Diseases of Livestock Research Unit, USDA/ARS/National Animal Disease Center, P.O. Box 70, 2300 Dayton Road, Ames, Iowa 50010, USA. kregiste@nadc.ars.usda.gov
Yersin
Andrew G
AG
D000818:Animals; D001483:Base Sequence; D001885:Bordetella Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D042482:Bordetella avium / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D005069:Evaluation Studies as Topic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D011110:Polymorphism, Genetic; D011201:Poultry Diseases / Q000175:diagnosis* / Q000382:microbiology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D014422:Turkeys / Q000382:microbiology
D017931:DNA Primers; D004269:DNA, Bacterial