An alkaline phosphatase-conjugated 27 - basc oligonucleotide probe was developed to detect the gene, bfPA, encoding the bundle-- forming pilus of enteropathogenic Escherichia coli (EPEC). The probe was proven to be hi...An alkaline phosphatase-conjugated 27 - basc oligonucleotide probe was developed to detect the gene, bfPA, encoding the bundle-- forming pilus of enteropathogenic Escherichia coli (EPEC). The probe was proven to be highly specific to EPEC, hybridizing only toEPEC strains, but not to Salmonella spp. Shigella spp., Vibrio spp., and other E. coli isolates. one hundred seventy eight E. colistrains belonging to traditional EPEC sergroups isolated from patients with diarrhoea were tested the probe, the prevalence rate of bfpAgene was 25. 28%. The frequency of the bfpA gene in relation to serogroups varied widely among EPEc The bfPA gene was morecommonly detected in strains of class l serogroups of EPEC(41.0% ) than in class I serogroups of EPEC(11.6% 6 % ). HEP- 2 cell adhesion assay and the fluorescent actin staining (FAS) test are 100% argreement, with all the localized adherence (LA) positive strainsproucing positive FAS activity. The results of bfpA probe hybridization were of 98.9% agreement with those obtained by using HEP- 2 cell adhesion assay and the FAS test. the sensitivity and specifity of the probe versus the FAS test and the HEP- 2 cell assay were95.7% and 100%, respectively. This probe was proven to be useful in identifying EPEC which carries the bfpA gene.展开更多
由寡核苷酸引导的体外诱变技术又称定向诱变技术,它包括下列步骤;克隆待诱变的DNA 至 phagemid;制备单链 DNA 模板;设计并合成特殊的诱变寡核苷酸(在寡核苷酸中,除其中少数几个待诱变的碱基外,其余均与单链 DNA 模板上的待定区域互补);...由寡核苷酸引导的体外诱变技术又称定向诱变技术,它包括下列步骤;克隆待诱变的DNA 至 phagemid;制备单链 DNA 模板;设计并合成特殊的诱变寡核苷酸(在寡核苷酸中,除其中少数几个待诱变的碱基外,其余均与单链 DNA 模板上的待定区域互补);合成双链 DNA。通过上述步骤,我们使 Stx-B 基因的 N 末端产生了新的 BamH Ⅰ位点,在 C 末端产生了新的Bgl Ⅱ识别序列,从而为 Stx-B 基因融合至 LamB 基因创造了条件。该法是遗传工程中不可缺少的重要手段之一。展开更多
文摘An alkaline phosphatase-conjugated 27 - basc oligonucleotide probe was developed to detect the gene, bfPA, encoding the bundle-- forming pilus of enteropathogenic Escherichia coli (EPEC). The probe was proven to be highly specific to EPEC, hybridizing only toEPEC strains, but not to Salmonella spp. Shigella spp., Vibrio spp., and other E. coli isolates. one hundred seventy eight E. colistrains belonging to traditional EPEC sergroups isolated from patients with diarrhoea were tested the probe, the prevalence rate of bfpAgene was 25. 28%. The frequency of the bfpA gene in relation to serogroups varied widely among EPEc The bfPA gene was morecommonly detected in strains of class l serogroups of EPEC(41.0% ) than in class I serogroups of EPEC(11.6% 6 % ). HEP- 2 cell adhesion assay and the fluorescent actin staining (FAS) test are 100% argreement, with all the localized adherence (LA) positive strainsproucing positive FAS activity. The results of bfpA probe hybridization were of 98.9% agreement with those obtained by using HEP- 2 cell adhesion assay and the FAS test. the sensitivity and specifity of the probe versus the FAS test and the HEP- 2 cell assay were95.7% and 100%, respectively. This probe was proven to be useful in identifying EPEC which carries the bfpA gene.
