IMMUNOMAGNETIC SEPARATION AND SOLID-PHASE DETECTION OF BORDETELLA-PERTUSSIS

In the present study, novel solid-phase methods mere used for both sam ple preparation and PCR detection of Bordetella pertussis. The sample preparation was performed by immunomagnetic separation with paramagnet ic beads coated with polyclonal antibodies directed toward the surface antigens of the bacteria. The precoated immunobeads were directly use d on nasopharyngeal aspirates to capture the bacteria on the solid sup port and were subsequently transferred to the PCR tube with no further manipulations, The region encompassing the pertussis toxin promoter w as analyzed to allow direct discrimination between the three major Bor detella species (B. pertussis, B. parapertussis, and B. bronchiseptica ). The resulting amplicons were captured on a second magnetic solid ph ase, allowing detection and restriction analysis of the target sequenc e. A colorimetric detection system based on a DNA binding fusion prote in enabled the use of standardized enzyme-linked immunosorbent format tests both for the detection of Bordetella spp. and for species evalua tion. When the optimized system was evaluated on 55 clinical aspirate samples, 21 of 22 (95%) culture-positive samples were positive by the system that we developed. In addition, two samples were positive by th e PCR-based assay, while the culture assay was negative. The implicati ons of these results are discussed.