Detection of multidrug resistance in Mycobacterium tuberculosis
Detection of multidrug resistance in Mycobacterium tuberculosis
复制标题
DOI：
10.1128/jcm.00750-06
复制
发表时间：
2007-01-01
影响因子：
9.4
通讯作者：
Kirikae, Teruo
中科院分区：
文献类型：
--
作者：
Sekiguchi, Jun-ichiro;Miyoshi-Akiyama, Tohru;Kirikae, Teruo
我们开发了一种基于DNA测序的方法来检测耐药结核分枝杆菌基因组中的突变。M.结核病是由基因组限制区域的突变引起的。采用PCR方法同时扩增利福平（RIF）rpoB、异烟肼（INH）katG和mabA（fabGl）-inhA启动子、乙胺丁醇（EMB）embB、吡嗪酰胺（PZA）pncA、链霉素（STR）rpsL和rrs、左氧氟沙星（levofloxacin）gyrA等8个与耐药相关的基因组区域，并测定其DNA序列。完成所有手术需要6.5小时。在检测的138株临床分离株中，55株对至少一种药物耐药。38株INH耐药株中有34株（89.5%）、28株RIF耐药株中有28株（100%）、18株EMB耐药株中有15株（83.3%）、30株STR耐药株中有18株（60%）、17株PZA耐药株中有17株（100%）存在特异性耐药相关突变。这些突变中有18个以前没有报道过。这些新的突变包括rpoB中的一个，katG中的八个，mabA-inh，4调节区中的一个，embB中的两个，pncA中的五个，以及rrs中的一个。大肠杆菌菌株单独表达的8个katG突变中的5个表现出过氧化氢酶和INH氧化活性的损失，携带任何5个pncA突变的菌株没有表现出吡嗪酰胺酶活性，表明这些突变分别与INH和PZA抗性相关。我们基于测序的方法也可用于检测结核病患者的结核分枝杆菌和筛选牛分枝杆菌的突变。结论：该方法可用于多重耐药支原体的快速检测。结核病和鉴定耐药M.结核
We developed a DNA sequencing-based method to detect mutations in the genome of drug-resistant Mycobacterium tuberculosis. Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome. Eight genome regions associated with drug resistance, including rpoB for rifampin (RIF), katG and the mabA (fabGl)-inhA promoter for isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR), and gyrA for levofloxacin, were amplified simultaneously by PCR, and the DNA sequences were determined. It took 6.5 h to complete all procedures. Among the 138 clinical isolates tested, 55 were resistant to at least one drug. Thirty-four of 38 INH-resistant isolates (89.5%), 28 of 28 RIF-resistant isolates (100%), 15 of 18 EMB-resistant isolates (83.3%), 18 of 30 STR-resistant isolates (60%), and 17 of 17 PZA-resistant isolates (100%) had mutations related to specific drug resistance. Eighteen of these mutations had not been reported previously. These novel mutations include one in rpoB, eight in katG, one in the mabA-inh,4 regulatory region, two in embB, five in pncA, and one in rrs. Escherichia coli isolates expressing individually five of the eight katG mutations showed loss of catalase and INH oxidation activities, and isolates carrying any of the five pncA mutations showed no pyrazinamidase activity, indicating that these mutations are associated with INH and PZA resistance, respectively. Our sequencing-based method was also useful for testing sputa from tuberculosis patients and for screening of mutations in Mycobacterium bovis. In conclusion, our new method is useful for rapid detection of multipie-drug-resistant M. tuberculosis and for identifying novel mutations in drug-resistant M. tuberculosis.