Rapid Detection of Multidrug-Resistant Mycobacterium tuberculosis by Use of Real-Time PCR and High-Resolution Melt Analysis
Rapid Detection of Multidrug-Resistant Mycobacterium tuberculosis by Use of Real-Time PCR and High-Resolution Melt Analysis
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DOI：
10.1128/jcm.00812-10
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发表时间：
2010-11-01
影响因子：
9.4
通讯作者：
Posey, James E.
中科院分区：
文献类型：
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作者：
Ramirez, Melissa V.;Cowart, Kelley C.;Posey, James E.
目前的研究描述了一种独特的实时荧光聚合酶链式反应检测结核分枝杆菌耐药突变的方法。RpoB的利福平耐药决定区(RRDR)、katG的特异区和inha启动子分别用于检测利福平(RIF)和异烟肼(INH)耐药。此外，通过针对IS6110插入元件，该方法被多重用于区分结核分枝杆菌复合体(MTC)和非结核分枝杆菌(NTM)菌株。采用实时荧光定量聚合酶链式反应(RT-PCR)后的高分辨熔融(HRM)分析方法对含有突变的结核分枝杆菌菌株进行鉴定，并使用锁定核酸(LNA)探针加强对含有特异性单核苷酸多态性(SNP)颠换突变的菌株的检测。应用该方法对252株结核分枝杆菌临床分离株进行筛选，其中RIF耐药株154株，异烟肼耐药株174株。在154株耐RIF的菌株中，148株也对异烟肼耐药，因此被归类为多药耐药(MDR)。该方法检测RIF耐药性的敏感性和特异性分别为91%和98%，检测异烟肼耐药性的敏感性和特异性分别为87%和100%。总体而言，该方法检测耐多药菌株的敏感性为85%，特异性为98%。这种方法提供了一种快速、可靠和廉价的方法来检测已知的导致结核分枝杆菌多药耐药的显性突变，并提供了几个优于当前基于分子和培养的技术的优点。
The current study describes the development of a unique real-time PCR assay for the detection of mutations conferring drug resistance in Mycobacterium tuberculosis. The rifampicin resistance determinant region (RRDR) of rpoB and specific regions of katG and the inhA promoter were targeted for the detection of rifampin (RIF) and isoniazid (INH) resistance, respectively. Additionally, this assay was multiplexed to discriminate Mycobacterium tuberculosis complex (MTC) strains from nontuberculous Mycobacteria (NTM) strains by targeting the IS6110 insertion element. High-resolution melting (HRM) analysis following real-time PCR was used to identify M. tuberculosis strains containing mutations at the targeted loci, and locked nucleic acid (LNA) probes were used to enhance the detection of strains containing specific single-nucleotide polymorphism (SNP) transversion mutations. This method was used to screen 252 M. tuberculosis clinical isolates, including 154 RIF-resistant strains and 174 INH-resistant strains based on the agar proportion method of drug susceptibility testing (DST). Of the 154 RIF-resistant strains, 148 were also resistant to INH and therefore classified as multidrug resistant (MDR). The assay demonstrated sensitivity and specificity of 91% and 98%, respectively, for the detection of RIF resistance and 87% and 100% for the detection of INH resistance. Overall, this assay showed a sensitivity of 85% and a specificity of 98% for the detection of MDR strains. This method provides a rapid, robust, and inexpensive way to detect the dominant mutations known to confer MDR in M. tuberculosis strains and offers several advantages over current molecular and culture-based techniques.