RESUMEN
To simplify detection procedures of DNA fragments resulting from PCR, we developed a colorimetric microplate hybridization assay. This format was used for the identification of Borrelia burgdorferi sensu lato, the causal agent of Lyme disease. The system relied on the use of a specific capture probe covalently linked to polystyrene plates and a specific polybiotinylated detection probe. DNA fragments, resulting from PCR and sandwiched between these two probes, were detected by enzymatic color development. The new detection format outperformed agarose gel electrophoresis of PCR products in sensitivity and specificity Moreover, in view of its rapidity and simplicity, the system proved appropriate for the routine diagnostic analysis of clinical specimens from Lyme disease patients. The proposed detection format can be adapted easily to other DNA targets and is suitable for automation.
Asunto(s)
Grupo Borrelia Burgdorferi/genética , Colorimetría , ADN Bacteriano/análisis , Hibridación de Ácido Nucleico/métodos , Biotina , Sondas de ADN , Humanos , Enfermedad de Lyme/líquido cefalorraquídeo , Enfermedad de Lyme/microbiología , Enfermedad de Lyme/orina , Reacción en Cadena de la Polimerasa , Poliestirenos , Sensibilidad y Especificidad , Piel/microbiología , Líquido Sinovial/microbiología , Moldes GenéticosAsunto(s)
Infecciones por Borrelia/genética , Grupo Borrelia Burgdorferi/genética , Borrelia/genética , Ixodes/microbiología , Enfermedad de Lyme/genética , Ninfa/microbiología , Animales , Infecciones por Borrelia/epidemiología , Infecciones por Borrelia/etiología , Variación Genética , Humanos , Enfermedad de Lyme/epidemiología , Enfermedad de Lyme/etiología , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Oligonucleotide primers based on Borrelia burgdorferi sensu lato ospA gene sequences have been designed for use in the PCR to type all (SL primers) or each (GI to GIII primers) of the B. burgdorferi sensu lato genospecies involved in Lyme disease. These genospecies-specific primers were then used in the PCR on 24 biological fluids collected from 18 neuroborreliosis patients. Among the samples tested, 20 contained DNA from Borrelia garinii, 11 contained DNA from B. burgdorferi sensu stricto, and 10 contained DNA from Borrelia afzelii. In toto, 10 patients appeared to have been infected by a single genospecies and 8 were infected by more than one Lyme disease-associated genospecies. Serum specimens from six patients were absorbed with heterologous antigens and tested by Western blotting (immunoblotting). In four cases, residual immunodetection revealed specific epitopes of genospecies also detected by PCR; in two of them, the concordant results indicated pluri-infection of the patients. In the other two cases, Western blotting showed specific antibodies for two genospecies of Borrelia, while PCR detected DNA from only one. In summary, the data underscored the relatively high prevalence of pluri-infections in Lyme disease and confirmed the association of B. garinii with neuroborreliosis.