Immunomagnetic separation and solid-phase detection of Bordetella pertussis.
AUTOR(ES)
Stark, M
RESUMO
In the present study, novel solid-phase methods were used for both sample preparation and PCR detection of Bordetella pertussis. The sample preparation was performed by immunomagnetic separation with paramagnetic beads coated with polyclonal antibodies directed toward the surface antigens of the bacteria. The precoated immunobeads were directly used on nasopharyngeal aspirates to capture the bacteria on the solid support and were subsequently transferred to the PCR tube with no further manipulations. The region encompassing the pertussis toxin promoter was analyzed to allow direct discrimination between the three major Bordetella species (B. pertussis, B. parapertussis, and B. bronchiseptica). The resulting amplicons were captured on a second magnetic solid phase, allowing detection and restriction analysis of the target sequence. A colorimetric detection system based on a DNA binding fusion protein enabled the use of standardized enzyme-linked immunosorbent format tests both for the detection of Bordetella spp. and for species evaluation. 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 the PCR-based assay, while the culture assay was negative. The implications of these results are discussed.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=228892Documentos Relacionados
- Sensitive Detection of Escherichia coli O157:H7 in Food and Water by Immunomagnetic Separation and Solid-Phase Laser Cytometry
- Solid-phase radioimmunoassay for the detection of rotavirus.
- Primer specific solid-phase detection of PCR products.
- Immobilization of microorganisms for detection by solid-phase immunoassays.
- Separation and purification of the hemagglutinins from Bordetella pertussis.