CDC researchers developed a real-time PCR assay targeting insertion sequence (IS481) and pertussis toxin subunit 1 (ptxS1) of Bordetella pertussis. This real-time nucleic acid assay offers rapid, sensitive, and quantitative results. The employed primers have been validated through extensive diagnostic testing of 41 Bordetella and 64 non-Bordetella clinical isolates. This technology can be used to diagnose and distinguish B. pertussis, B. parapertussis and B. homesii, the three most common Bordetella human upper respiratory pathogens. A standalone assay or multi-faceted kit may be used.