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 PCRbased assay, while the culture assay was negative. The implications of these results are discussed.

We isolated and characterized a Vibrio cholerae O139 Bengal-specific DNA region by arbitrary PCR. The fragment contains open reading frames encoding two potential glycosyltransferases possibly involved in capsular polysaccharide or lipopolysaccharide biosynthesis. In order to evaluate the possibility that this region could be used for the specific detection of V. cholerae O139 Bengal, a PCR system was established. The specificity and sensitivity of the PCR were investigated by analyzing 240 strains within the family Vibrionaceae and 178 strains of other gram-negative bacteria. All V. cholerae O139 Bengal strains tested were positive, and none of the 384 control strains were amplified. The sensitivity of the assay was 10 2 CFU/ml.

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An integrated diagnostic method for colorimetric detection of polymerase chain reaction (PCR) products in a microtitre format is described. The amplified material is labelled by using modified PCR primers introducing a biotin molecule and a lac operator handle. Positive samples are identified, after immobilization onto streptavidin-coated microtitre wells, using a reporter fusion protein consisting of the Escherichia coli lac repressor and beta-galactosidase. The analysis of the PCR products is thus, from a practical point of view, identical with the enzyme-linked immunosorbent assay (ELISA) using the enzymatic activity of beta-galactosidase. Here, we show that the assay can be used for rapid and simple detection of Chlamydia trachomatis in an automated format. The reported analysis of 90 urethral patient samples, using this microtitre assay, indicates that this technique is more sensitive and more specific than culture technique.