RESUMO
SUMMARY: A highly sensitive assay for rapidly screening-out Mycobacterium bovis in contaminated samples was developed based on electrochemical genosensing. The assay consists of specific amplification and double-tagging of the IS6110 fragment, highly related to M. bovis, followed by electrochemical detection of the amplified product. PCR amplification was carried out using a labeled set of primers and resulted in a amplicon tagged at each terminus with both biotin and digoxigenin. Two different electrochemical platforms for the detection of the double-tagged amplicon were evaluated: (i) an avidin biocomposite (Av-GEB) and (ii) a magneto sensor (m-GEC) combined with streptavidin magnetic beads. In both cases, the double- tagged amplicon was immobilized through its biotinylated end and electrochemically detected, using an antiDig-HRP conjugate, through its digoxigenin end. The assay was determined to be highly sensitive, based on the detection of 620 and 10 fmol of PCR amplicon using the Av-GEB and m-GEC strategies, respectively. Moreover, the m-GEC assay showed promising features for the detection of M. bovis on dairy farms by screening for the presence of the bacterium's DNA in milk samples. The obtained results are discussed and compared with respect to those of inter-laboratory PCR assays and tuberculin skin testing.
Assuntos
Técnicas Bacteriológicas/métodos , Técnicas Eletroquímicas/métodos , Programas de Rastreamento/métodos , Leite/microbiologia , Mycobacterium bovis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , Biotina/química , Primers do DNA/química , Primers do DNA/genética , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Digoxigenina/química , Mycobacterium bovis/genética , Sensibilidade e Especificidade , Coloração e Rotulagem/métodosRESUMO
A highly sensitive assay for rapidly screening-out Mycobacterium bovis in contaminated samples was developed based on electrochemical genosensing. The assay consists of specific amplification and double-tagging of the IS6110 fragment, highly related to M. bovis, followed by electrochemical detection of the amplified product. PCR amplification was carried out using a labeled set of primers and resulted in a amplicon tagged at each terminus with both biotin and digoxigenin. Two different electrochemical platforms for the detection of the double-tagged amplicon were evaluated: (i) an avidin biocomposite (Av-GEB) and (ii) a magneto sensor (m-GEC) combined with streptavidin magnetic beads. In both cases, the double- tagged amplicon was immobilized through its biotinylated end and electrochemically detected, using an antiDig-HRP conjugate, through its digoxigenin end. The assay was determined to be highly sensitive, based on the detection of 620 and 10 fmol of PCR amplicon using the Av-GEB and m-GEC strategies, respectively. Moreover, the m-GEC assay showed promising features for the detection of M. bovis on dairy farms by screening for the presence of the bacterium's DNA in milk samples. The obtained results are discussed and compared with respect to those of inter-laboratory PCR assays and tuberculin skin testing (AU)
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Assuntos
Animais , Técnicas Bacteriológicas/métodos , Programas de Rastreamento/métodos , Leite , Mycobacterium bovis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Mycobacterium bovis/genética , Sensibilidade e Especificidade , Coloração e Rotulagem/métodosRESUMO
La enfermedad celíaca se presenta con una gran heterogeneidad: formas sintomáticas, silentes y latentes. La determinación de anticuerpos antigliadina y antiendomisio es útil para definir a qué pacientes realizar biopsia de intestino delgado y para el seguimiento de los celíacos ya conocidos. Estas técnicas fueron incorporadas en 1994 en el Centro Hospitalario Pereira Rossell. Objetivo: conocer la sensibilidad y especificidad de los anticuerpos antigliadina (IgG) y antiendomisio (IgA) para el diagnóstico de enfermedad celíaca, determinados por técnica de inmunofluorescencia en la Sección de Inmunologia del Laboratorio Central del Centro Hospitalario Pereira Rossell y aplicados a población hospitalaria. Material y método: se evaluaron los resultados de la serología tomando como patrón de oro la anatomía patológica de la biopsia de intestino delgado. Para el diagnóstico de enfermedad celíaca se siguieron los criterios de 1990 de la Sociedad Europea de Gastroenterología Pediátrica y Nutrición. Se evaluó la serología de 65 niños que recibían gluten al momento de la extracción de sangre: 50 celíacos y 15 no celíacos (con biopsia normal). Resultados: la sensibilidad y la especificidad fueron de 94 por ciento y 80 por ciento para los anticuerpos antigliadina y de 94 por ciento y 93 por ciento para los antiendomisio respectivamente. Comentarios: estos resultados son similares a los comunicados por los mejores laboratorios a nivel internacional
