RESUMEN
A simple, sensitive and specific colourimetric hybridisation method for the detection of HCMV DNA in clinical specimens is described. This method combines a PCR assay with a sensitive sandwich hybridisation assay. It relies on the use of a specific capture probe linked covalently to polystyrene microplates and a specific polybiotinylated detection probe. Amplified DNA fragments, sandwiched between these two probes, are detected by an enzymatic colour reaction. This PCR-based colourimetric hybridisation method was compared with other known HCMV detection methods. Clinical specimens (n = 145, corresponding to 106 patients) were tested by both a nested PCR assay and this colourimetric hybridisation method; and by either the culture method or the pp65 antigenaemia test depending on the type of sample used. The results showed that the PCR-based hybridisation method has a specificity similar to tissue culture, known as the conventional gold standard method, and could be used for the examination of the clinical specimens.
Asunto(s)
Citomegalovirus/genética , ADN Viral/aislamiento & purificación , Hibridación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa/métodos , Adolescente , Adulto , Anciano , Líquido Amniótico/química , Líquido Amniótico/virología , Niño , Preescolar , ADN Viral/sangre , ADN Viral/líquido cefalorraquídeo , ADN Viral/orina , Endopeptidasa K/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Placenta/química , Sensibilidad y Especificidad , Cuerpo Vítreo/químicaRESUMEN
A colorimetric microplate hybridization assay was developed previously to simplify detection procedures of DNA fragments resulting from polymerase chain reactions (PCR). This format has now been adapted for the simultaneous detection and identification of three human papillomavirus (HPV), types 16, 18 and 33, associated frequently with cervical cancer. This post-PCR detection system uses three type-specific capture oligonucleotides linked covalently to a single microplate well and three type-specific multibiotinylated oligonucleotidic probes for detection. It therefore offers a double specificity; the first is conferred by pairs of primers, specific of each type of virus tested, and the second, by the sets of capture and detection probes which are complementary to internal regions of the amplified DNA fragments. The detection format outperformed agarose gel electrophoresis of amplified DNA products in sensitivity and specificity. The rapidity and simplicity of this hybridisation system would justify its use in routine diagnostic examination of cervical specimens (smears and biopsies).
Asunto(s)
Colorimetría/métodos , ADN Viral/análisis , Proteínas de Unión al ADN , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/virología , Reacción en Cadena de la Polimerasa/métodos , Infecciones Tumorales por Virus/virología , Estudios de Evaluación como Asunto , Femenino , Humanos , Hibridación de Ácido Nucleico , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Proteínas E7 de Papillomavirus , Infecciones por Papillomavirus/patología , Sensibilidad y Especificidad , Infecciones Tumorales por Virus/patologíaRESUMEN
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.