Digoxigenin labelled DNA probes for rapid detection of enterotoxigenic, enteropathogenic and Vero cytotoxin producing Escherichia coli in faecal samples

Frequency of enterovirulentEscherichia coli in diarrhoeal disease in The Netherlands

“…Probes were multiplied using the polymerase chain reaction. Primers used for the generation of the probes, length, composition of the PCR reaction mixture, appropriate hybridization temperatures and detection methods were as previously described (9). Probes were also labelled during the polymerase chain reaction by adding digoxigenin-dUTP (Boehringer, FRG) to the PCR reaction mixture (9).…”

“…After overnight incubation at 30 *C bacteria were replica-plated onto Zprobe filters (Bio-Rad Laboratories, USA). Bacteria were lysed and DNA fixed onto the fiiters as described before (9).…”

“…DNA hybridization technology has facilitated the detection of Escherichia coli strains that cause diarrhoea. In a previous study, we developed a simple and sensitive hybridization assay to detect EPEC, VTEC and ETEC directly in faecal specimen using a set of five non-radioactively labelled DNA fragment probes (9). This method allows epidemiologic studies on a relatively large scale.…”

Principales características y diagnóstico de los grupos patógenos de Escherichia coli

“…En esta prueba se requiere una área destinada a la preparación de las muestras para PCR y otra sólo para productos de PCR, ya que se debe evitar posibles contaminaciones El cuadro V presenta algunas secuencias de iniciadores y sondas empleadas en el diagnóstico. 4,61 En el InDRE desde 1993 se empezó la estandarización de métodos de biología molecular como "colony blot" y PCR los cuales desde 1996 se emplean con fines diagnósticos y como apoyo a la vigilancia epidemiológica, para la caracterización de E. coli patógena aislada de casos de diarrea y cuyos resultados han sido presentados en diversas reuniones científicas. 62 Los resultados obtenidos indican que el grupo más frecuente es el enterotoxigénico (48%), seguido por el enteroinvasivo (9%), el enteropatógeno (4%) y el enterohemorrágico (1%), del cual sólo se han aislado cepas no O157:H7.…”

Detection of toxigenicClostridium difficile in fecal samples by colony blot hybridization

“…The part of the gel containing the probe was excised and the probe was recovered by electro-elution(18). Briefly the PCR mixture (100/al) contained 50 mM KC1, 10 mM Tris-HC1 (pH 8.3), 1.5 mM MgCI2, 0.01% gelatine, 90 tam dTTP, 10 /aM digoxigenin-dUTP (Boehringer, Germany), 100 }aM each of dATP, dCTP and dCTP, 0.5/aM of each primer and 1.25 U Taqpolymerase.…”