Development and evaluation of dual-target real-time polymerase chain reaction assays to detect Bordetella spp.

Novel, highly specific, and sensitive real-time polymerase chain reaction (PCR) assays using 2 targets, insertion sequence (IS481) and pertussis toxin subunit 1 (ptxS1), were developed to detect Bordetella pertussis and to differentiate between relevant Bordetella spp. Sixty-four non-Bordetella isolates were negative by both assays, demonstrating the specificity of the assays. B. pertussis, Bordetella parapertussis, and Bordetella holmesii isolates were specifically identified using the assays. The lower limit of detection was less than 10 genomic equivalents per reaction for the IS481 and ptxS1 assays. These assays were evaluated using 145 human clinical specimens obtained during cough-illness outbreak investigations, and PCR results were compared with Bordetella spp. culture results. Twenty-seven (18.6%) specimens had late positive cycle threshold (Ct) values (35 ≤ Ct < 40) using the IS481 assay with corresponding negative results using the ptxS1 assay and culture and were considered indeterminate. Guidelines for use of PCR testing and interpretation of results during cough-illness outbreaks are discussed.