Molecular detection of Bordetella pertussis DNA is a sensitive and specific method for the rapid diagnosis of pertussis. In this study, a new molecular assay for the detection and differentiation of Bordetella spp. based on automated DNA extraction and real-time PCR was evaluated. The analytical sensitivity of the new assay was determined by Probit analysis of serial dilutions of both cloned PCR products IS481 and IS1001 and cell suspensions of B. pertussis, B. parapertussis, and B. bronchiseptica. The specificity was analyzed by testing a number of pathogens producing respiratory infections. Moreover, a total of 92 clinical samples were investigated. The results were compared to those obtained by an in-house assay based on manual DNA extraction, followed by real-time PCR and detection of IS481. The analytical sensitivity of the new assay for the detection of IS481 and IS1001 was determined to be 2.2 and 1.2 genome equivalents/l, respectively. The analytical sensitivity for the detection of B. pertussis, B. parapertussis, and B. bronchiseptica was determined to be 1.6, 1.0, and 2.7 genome equivalents/l, respectively. When clinical specimens were tested with the new assay, 46 of 92 were found to be positive for Bordetella DNA. With the in-house assay, 45 samples tested positive. The new molecular assay proved to be suitable for the rapid diagnosis of pertussis in the routine diagnostic laboratory.Bordetella pertussis is the agent responsible for producing pertussis, also called whooping cough. B. parapertussis, B. bronchiseptica, and B. holmesii may also cause a pertussis-like respiratory disease but with a milder clinical course (7). Pertussis occurs worldwide, with an increased incidence among young unvaccinated infants. Neonates and elderly patients may show an atypical course of disease (2, 4). A rapid and reliable diagnostic method is thus essential for appropriate treatment and prophylaxis. Culture has been considered the gold standard for detection of B. pertussis but often lacks sensitivity, and a minimum of 4 days may be required to obtain results (1).Today, PCR has been shown to be a sensitive and specific method for the rapid diagnosis of pertussis (3,6,11). In the Bordetella genome, there are repetitive insertion sequences present in a high copy number that may be useful for the amplification of different bacterial strains. Insertion sequences include IS481 in B. pertussis, B. holmesii, and, occasionally B. bronchiseptica, and IS1001 in B. parapertussis and, occasionally, B. bronchiseptica and B. holmesii. These sequences have been reported as target sequences in several PCR protocols (5,10,13,15).In the present study, the new artus Bordetella LC PCR Kit (QIAGEN Hamburg GmbH, Germany), which includes a multiplex PCR system consisting of four independent PCRs for qualitative detection and differentiation of B. pertussis, B. parapertussis, B. bronchiseptica, and the heterologous internal control (IC), was evaluated. After we tested these methods for analytical sensitivity and specificity, clinical speci...
