Detection of rifampin resistance in Mycobacterium tuberculosis in a single tube with molecular beacons
Detection of rifampin resistance in Mycobacterium tuberculosis in a single tube with molecular beacons
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DOI：
10.1128/jcm.39.11.4131-4137.2001
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发表时间：
2001-11-01
影响因子：
9.4
通讯作者：
Alland, D
中科院分区：
文献类型：
--
作者：
El-Hajj, HH;Marras, SAE;Alland, D
由于结核杆菌生长缓慢，目前确定结核分枝杆菌抗生素敏感性的临床测定需要数周才能完成。在这里，我们展示了一种极其灵敏的单管 PCR 测定，该测定只需不到 3 小时，就能可靠地识别直接从痰中提取的 DNA 中的利福平耐药结核分枝杆菌。 95% 与利福平耐药相关的突变发生在细菌 RNA 聚合酶基因 rpoB 的 81 个核心区域。该区域内发生的所有突变都会导致利福平耐药。该测定使用称为分子信标的新型核酸杂交探针。在同一反应中使用五种不同的探针，每种探针与利福平敏感杆菌的 rpoB 基因内的不同靶序列完全互补，并且每种探针都用不同颜色的荧光团标记。它们的靶序列一起涵盖了整个核心区域。 PCR 扩增过程中所有五种荧光颜色的产生表明存在对利福平敏感的结核分枝杆菌。核心区域中任何突变的存在都会阻止分子信标之一的结合，从而导致五种荧光颜色之一的缺失。当测试 148 个已知对利福平敏感的结核分枝杆菌临床分离株时，在 65 个利福平耐药分离株中的 63 个中检测到与利福平耐药相关的突变，而在 83 个利福平敏感分离株中没有发现任何突变。当对 11 名耐利福平结核病感染者的痰液中直接提取的 DNA 进行检测时，所有样本均检测到了突变。这种快速检测的使用应该能够在临床环境中及早发现和治疗耐药结核病。
Current clinical assays for determining antibiotic susceptibility in Mycobacterium tuberculosis require many weeks to complete due to the slow growth of the bacilli. Here we demonstrate an extremely sensitive single-tube PCR assay that takes less than 3 h and reliably identifies rifampin-resistant M. tuberculosis in DNA extracted directly from sputum. Ninety-five percent of mutations associated with rifampin resistance occur in an 81-by core region of the bacterial RNA polymerase gene, rpoB. All mutations that occur within this region result in rifampin resistance. The assay uses novel nucleic acid hybridization probes called molecular beacons. Five different probes are used in the same reaction, each perfectly complementary to a different target sequence within the rpoB gene of rifampin-susceptible bacilli and each labeled with a differently colored fluorophore. Together, their target sequences encompass the entire core region. The generation of all five fluorescent colors during PCR amplification indicates that rifampin-susceptible M. tuberculosis is present. The presence of any mutation in the core region prevents the binding of one of the molecular beacons, resulting in the absence of one of the five fluorescent colors. When 148 M. tuberculosis clinical isolates of known susceptibility to rifampin were tested, mutations associated with rifampin resistance were detected in 63 of the 65 rifampin-resistant isolates, and no mutations were found in any of the 83 rifampin-susceptible isolates. When DNA extracted directly from the sputum of 11 patients infected with rifampin-resistant tuberculosis was tested, mutations were detected in all of the samples. The use of this rapid assay should enable early detection and treatment of drug-resistant tuberculosis in clinical settings.