目的 研究制备 nor A基因介导的金黄色葡萄球菌对氟喹诺酮类药物的耐药机制的 Dig- nor A基因探针。方法 采用聚合酶链反应 (PCR)制备 Dig- nor A基因探针。结果 PCR法制备 Dig- nor A基因探针简便易行 ,可在较短时间内获得大量的探...目的 研究制备 nor A基因介导的金黄色葡萄球菌对氟喹诺酮类药物的耐药机制的 Dig- nor A基因探针。方法 采用聚合酶链反应 (PCR)制备 Dig- nor A基因探针。结果 PCR法制备 Dig- nor A基因探针简便易行 ,可在较短时间内获得大量的探针 ,所得探针有较高的敏感性 ;Dig- nor A基因探针安全、易操作 ,标记探针可长期保存。结论 为进一步研究 nor展开更多
按金黄色葡萄球菌多重耐药转运蛋白NorA的编码序列设计引物,以金黄色葡萄球菌基因组DNA为模板,扩增出norA基因中1 155 bp cDNA片段,将所得片段与pMD18-T载体连接,转化到感受态大肠杆菌JM109中,成功地筛选到阳性克隆,其质粒测序结果与文...按金黄色葡萄球菌多重耐药转运蛋白NorA的编码序列设计引物,以金黄色葡萄球菌基因组DNA为模板,扩增出norA基因中1 155 bp cDNA片段,将所得片段与pMD18-T载体连接,转化到感受态大肠杆菌JM109中,成功地筛选到阳性克隆,其质粒测序结果与文献报道一致。从阳性克隆中提取质粒,经NdeⅠ和XhoⅠ酶切,回收1 155 bp目的片段,定向克隆到pET-28a(+)表达载体中,转化感受态大肠杆菌DH5α,提取质粒,再次转化到BL21(DE3)中,成功地筛选到阳性克隆。经IPTG诱导阳性菌,通过SDS-PAGE检测出norA基因的表达。展开更多
AIM： Methicillin-resistant Staphylococcus aureus （MRSA） is a pathogenic bacterium that causes both hospital- and community-acquired infections, and for which single-drug treatments are becoming less efficient. Rhiz...AIM： Methicillin-resistant Staphylococcus aureus （MRSA） is a pathogenic bacterium that causes both hospital- and community-acquired infections, and for which single-drug treatments are becoming less efficient. Rhizoma coptidis has been used for more than two thousand years in China to treat diarrhea, fever, and jaundice. In this study, the anti-MRSA activity of Rhizoma coptidis is examined and its effective components sought. METHODS： The mecA and norA genes were determined by PCR amplification and sequencing. Drug susceptibility of Staphylococcus aureus ATCC43300 was performed using the VITEK2 compact system. The chemical fingerprint of Rhizoma coptidis was investigated using HPLC and preparative liquid chromatography, and the anti-MRSA activity was determined using an improved broth microdilution method. RESULTS： The drug susceptibility test revealed that the penicillin-binding protein phenotype of the strain changed in comparison to penicillin-sensitive Staphylococcus aureus. Ten batches of Rhizoma coptidis showed anti-MRSA activity on the norA-negative Staphylococcus aureus strain, as well as the strain that contained a norA gene. The spectrum-effect relationship revealed that the berberine alkaloids were the effective components, within which berberine, coptisine, palmatine, epiberberine,, and jatrorrhizine were the major components. CONCLUSION： This study lays a foundation for in vivo studies of Rhizoma eoptidis and for the development of multi-component drugs.展开更多
文摘目的 研究制备 nor A基因介导的金黄色葡萄球菌对氟喹诺酮类药物的耐药机制的 Dig- nor A基因探针。方法 采用聚合酶链反应 (PCR)制备 Dig- nor A基因探针。结果 PCR法制备 Dig- nor A基因探针简便易行 ,可在较短时间内获得大量的探针 ,所得探针有较高的敏感性 ;Dig- nor A基因探针安全、易操作 ,标记探针可长期保存。结论 为进一步研究 nor
基金supported by the National Natural Science Foundation of China(No.81073043)
文摘AIM： Methicillin-resistant Staphylococcus aureus （MRSA） is a pathogenic bacterium that causes both hospital- and community-acquired infections, and for which single-drug treatments are becoming less efficient. Rhizoma coptidis has been used for more than two thousand years in China to treat diarrhea, fever, and jaundice. In this study, the anti-MRSA activity of Rhizoma coptidis is examined and its effective components sought. METHODS： The mecA and norA genes were determined by PCR amplification and sequencing. Drug susceptibility of Staphylococcus aureus ATCC43300 was performed using the VITEK2 compact system. The chemical fingerprint of Rhizoma coptidis was investigated using HPLC and preparative liquid chromatography, and the anti-MRSA activity was determined using an improved broth microdilution method. RESULTS： The drug susceptibility test revealed that the penicillin-binding protein phenotype of the strain changed in comparison to penicillin-sensitive Staphylococcus aureus. Ten batches of Rhizoma coptidis showed anti-MRSA activity on the norA-negative Staphylococcus aureus strain, as well as the strain that contained a norA gene. The spectrum-effect relationship revealed that the berberine alkaloids were the effective components, within which berberine, coptisine, palmatine, epiberberine,, and jatrorrhizine were the major components. CONCLUSION： This study lays a foundation for in vivo studies of Rhizoma eoptidis and for the development of multi-component drugs.
