Objective:To present an integrated molecular biology dedicated system for tuberculosis diagnosis.Methods:One hundred and five sputum specimens from patients strongly suspected by clinical parameters of tuberculosis we...Objective:To present an integrated molecular biology dedicated system for tuberculosis diagnosis.Methods:One hundred and five sputum specimens from patients strongly suspected by clinical parameters of tuberculosis were studied by Ziehl-Neelsen staining,by cultivation on solid medium and by a balanced hemincsted fluorometric PCR system(Orange C3TB) that could preserve worker safety and produce a rather pure material free of potential inhibitors. DNA amplification was performed in a low cost tuberculosis termocycler-fluorotneter.Produced double stranded DNA was flurometrically detected.The whole reaction was conducted in one single tube which would not be opened after adding the processed sample in order to minimize the risk of cross contamination with amplicons.Results:The assay was able to delect 30 bacillus per sample mL with 99.8%interassay variation coefficient.PCR was positive in 23(21.9%) tested samples(21 of them were smear negative).In our study it showed a preliminary sensitivity of 94.5%for sputum and an overall specificity of 98.7%.Conclusions:Total run time of the test is 4 h with 2.5 real working time.All PCR positive samples are also positive by microbiological culture and clinical criteria.Results show that it could be a very useful tool to increase detection efficiency of tuberculosis disease in low bacilus load samples.Furthermore,its low cost and friendly using make it feasible to run in poor regions.展开更多
目的构建结核杆菌低分子质量抗原M tb8.4基因的原核表达重组质粒,为其蛋白质的表达及免疫学研究打下基础。方法PC R扩增目的基因,克隆到质粒pET-H is T7启动子的下游,转化大肠杆菌T O P10F',进行重组子的筛选、鉴定。结果M tb8.4基...目的构建结核杆菌低分子质量抗原M tb8.4基因的原核表达重组质粒,为其蛋白质的表达及免疫学研究打下基础。方法PC R扩增目的基因,克隆到质粒pET-H is T7启动子的下游,转化大肠杆菌T O P10F',进行重组子的筛选、鉴定。结果M tb8.4基因正向插入表达载体中,测序证实具有正确的读码框,基因序列与文献报道一致。结论重组原核表达质粒pET-H is-M tb8.4构建成功。展开更多
文摘Objective:To present an integrated molecular biology dedicated system for tuberculosis diagnosis.Methods:One hundred and five sputum specimens from patients strongly suspected by clinical parameters of tuberculosis were studied by Ziehl-Neelsen staining,by cultivation on solid medium and by a balanced hemincsted fluorometric PCR system(Orange C3TB) that could preserve worker safety and produce a rather pure material free of potential inhibitors. DNA amplification was performed in a low cost tuberculosis termocycler-fluorotneter.Produced double stranded DNA was flurometrically detected.The whole reaction was conducted in one single tube which would not be opened after adding the processed sample in order to minimize the risk of cross contamination with amplicons.Results:The assay was able to delect 30 bacillus per sample mL with 99.8%interassay variation coefficient.PCR was positive in 23(21.9%) tested samples(21 of them were smear negative).In our study it showed a preliminary sensitivity of 94.5%for sputum and an overall specificity of 98.7%.Conclusions:Total run time of the test is 4 h with 2.5 real working time.All PCR positive samples are also positive by microbiological culture and clinical criteria.Results show that it could be a very useful tool to increase detection efficiency of tuberculosis disease in low bacilus load samples.Furthermore,its low cost and friendly using make it feasible to run in poor regions.
文摘目的构建结核杆菌低分子质量抗原M tb8.4基因的原核表达重组质粒,为其蛋白质的表达及免疫学研究打下基础。方法PC R扩增目的基因,克隆到质粒pET-H is T7启动子的下游,转化大肠杆菌T O P10F',进行重组子的筛选、鉴定。结果M tb8.4基因正向插入表达载体中,测序证实具有正确的读码框,基因序列与文献报道一致。结论重组原核表达质粒pET-H is-M tb8.4构建成功。
