A novel multi-target real-time PCR assay for the rapid diagnosis of Bordetella species in clinical specimens.

Tatti KM, Sparks KN, Boney KO, Tondella ML.

Source

Centers for Disease Control and Prevention, National Center for Immunization and Respiratory Diseases, Division of Bacterial Diseases, Meningitis and Vaccine Preventable Diseases Branch, Atlanta, Georgia 30333.

Abstract

A novel multi-target real-time PCR (R-PCR) assay for the rapid identification and speciation of Bordetella pertussis, B. parapertussis, and B. holmesii was developed using multi-copy insertion sequences (IS) in combination with the pertussis toxin subunit S1 (ptxS1) singleplex assay. The R-PCR targets for the multiplex assay include IS481 commonly found in B. pertussis and B. holmesii, IS1001 of B. parapertussis, and the IS1001-like sequence of B. holmesii. Overall, 402 Bordetella spp. and 66 non-Bordetella spp. isolates were tested in the multi-target assay. Cross-reactivity was found only with 5 B. bronchiseptica isolates which were positive with IS1001 of B. parapertussis. The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format which was approximately 1 genomic equivalent per reaction for all targets. A total of 197 human clinical specimens obtained during cough-illness outbreak investigations were used to evaluate the multi-target R-PCR assay. The multiplex assay results from 87 clinical specimens were compared to the individual R-PCR assays and culture. The multi-target assay is useful as a diagnostic tool to confirm B. pertussis infections and to rapidly identify other Bordetella species. In conclusion, the use of this multi-target R-PCR approach increases specificity, while decreasing the amount of time, reagents, and specimen necessary for R-PCR reactions used for accurate diagnosis of pertussis-like illness.