【作者】 陆宇；

【导师】 朱莉贞； 段连山； 潘毓萱；

【作者基本信息】 北京市结核病胸部肿瘤研究所 ， 2003， 博士

【摘要】 目的 探讨结核分枝杆菌mRNA作为活菌检测分子标志的可行性，评价应用mRNA定量法进行结核分枝杆菌利福平敏感性分析和肺结核患者化疗反应监测的价值。方法 （1）采用定量RT-PCR方法测定结核分枝杆菌H₃₇R_v在利福平、异烟肼、乙胺丁醇、链霉素、氧氟沙星处理后24h、48h、72h 85B mRNA表达水平的变化。（2）应用定量RT-PCR方法检测53株结核分枝杆菌临床分离株对利福平的敏感性。（3）应用定量RT-PCR方法对15例初治肺结核患者应用标准短程化疗方案治疗过程中痰标本mRNA的系列监测。结果 （1）10μg/ml利福平处理24h后结核分枝杆菌85BmRNA下降到无药对照的0.02％，其他药物处理分别下降到无药对照的1％～10％，各药处理72h后85BmRNA均下降到1％以下，与菌落形成单位（CFU）呈平行关系。（2）53株结核分枝杆菌临床分离株mRNA定量法测定：利福平1μg/ml存在以1％和10％为耐药临界比例时与绝对浓度法的符合率分别为：69.8％、81.1％，利福平2μg/ml存在时符合率分别为84.9％、92.5％，利福平4μg/ml存在符合率是92.5％、92.5％。与绝对浓度法相比，mRNA定量法的敏感度在90％以上，特异度在2μg/ml利福平、以10％为临界比例时为87.5％。（3）结核分枝杆菌85BmRNA在15例肺结核患者治疗的2d快速下降，治疗14d时12例检测阴性，治疗60d时全部阴性。结论 （1）mRNA表达水平在24h内反映出含药与无药的区别，是检测和判定结核分枝杆菌“死”“活”的分子标志物。（2）mRNA定量法用于结核分枝杆菌药物敏感性分析具有快速、可定量等优点，建议以利福平2μg/ml作用24h后下降到无药对照的10％以下为利福平敏感，利福平2μg/ml作用24h未下降到无药对照的10％为利福平耐药。（3）结核分枝杆菌mRNA在接受治疗的肺结核患者的痰标本中快速下降，是活菌数量下降的反映，痰标本中mRNA的变化可作为快速评价化疗反应的分子指标。更多还原

【Abstract】 Objective To study whether quantitative analysis of Mycobacterium tuberculosis mRNA could be used to assess bacterial viability, to evaluate the significance of susceptibility of M. tuberculosis strains to rifampin and monitoring response to chemotherapy using by quantitative analysis of M. tuberculosis mRNA.Methods (1) Compared the levels of mRNA after 24, 48, 72 hours that M. tuberculosis H37Rv were treated with no drugs, rifampin isoniazid, ethambutol. streptomycin and ofloxacin. The levels of mRNA were determined by quantitative RT-PCR. (2) 29 rifampin resistant strains and 23 rifampin sensitive strains were test their susceptibility to rifampin by quantitative analysis of M. tuberculosis mRNA. (3) Quantitative measurement of sputum M. tuberculosis mRNA from 15 smear-positive pulmonary tuberculosis patients receiving an optimal drug treatment regimens. Results (1) Exposure of M. tuberculosis H37Rv to rifampin for 24h reduced the levels of 85BmRNA to 0.02%, exposure to other drugs for 24h reduced to 1%-10%, for 72h reduced to < 1%. (2) The agreement of quantitative analysis of M. tuberculosis mRNA with absolute concentration method was 69.8%, 84.9%, 92.5% respectability when rifampin 1μg/ml, 2μg/ml, 4μg/ml and 1% proportion was, 81.1%, 92.5%, 92.5% when 10% proportion was.Compared with absolute concentration method , the sensitivity of quantitative analysis of M. tuberculosis mRNA was >90%,the specialty was 87.5% when rifampin 2μg/ml and 10% proportion was.(3) The rapid decline of mRNA in sputum during the first two days of effective treatment,by day 14 of treatment , 12 of 15 subjects had no detectable mRNA ,by day 60 of treatment ,all subjects had no detectable mRNA in their sputum.Conclusions (1) The levels of mRNA were shown the differences exposure to drugs with no drug , and nearly parallel with CFU. mRNA is a marker for viability of M.tuberculosis. (2) Quantitative analysis of M. tuberculosis mRNA was a rapid, sensitive method in rifampin resistance screening , and showed mRNA declined to 10% of control when rifampin 2μg/ml was as rifampin- sensitive strains, otherwise as rifampin-resistant strains . (3) The rapid disappearance of M.tuberculosis mRNA from sputum suggests that it is a good indicator of microbial viability and a useful marker for rapid assessment of response to chemotherapy.更多还原