Bentaleb El Mehdi, El Messaoudi My Driss, Abid Mohammed, Messaoudi Malika, Yetisen Ali K, Sefrioui Hassan, Amzazi Saaïd, Ait Benhassou Hassan
Medical Biotechnology Center, Moroccan Foundation for Advanced Science, Innovation and Research (MAScIR), Rabat Design Center, Avenue Mohamed El Jazouli - Madinat Al Irfane, 10100, Rabat, Morocco.
Laboratory of Biochemistry and Immunology, Faculty of Sciences, Mohammed V University, Rabat, Morocco.
BMC Infect Dis. 2017 Aug 7;17(1):548. doi: 10.1186/s12879-017-2666-4.
Rapid diagnosis of drug resistance in tuberculosis (TB) is pivotal for the timely initiation of effective antibiotic treatment to prevent the spread of drug-resistant strains. The development of low-cost, rapid and robust methods for drug-resistant TB detection is highly desirable for resource-limited settings.
We report the use of an in house plasmid-based quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis for the detection of mutations related to rifampicin-resistant Mycobacterium tuberculosis (MTB) in clinical isolates from Moroccan patients. Five recombinant plasmids containing predominant mutations (S531L, S531W, H526Y and D516V) and the wild-type sequence of the Rifampicin Resistance-Determining Region (RRDR) have been used as controls to screen 45 rifampicin-resistant and 22 rifampicin-susceptible MTB isolates.
The sensitivity and the specificity of the qPCR-HRM analysis were 88.8% and 100% respectively as compared to rifampicin Drug Susceptibility Testing (DST). The results of qPCR-HRM and DNA sequencing had a concordance of 100%.
Our qPCR-HRM assay is a sensitive, accurate and cost-effective assay for the high-throughput screening of mutation-based drug resistance in TB reference laboratories.
结核病（TB）耐药性的快速诊断对于及时启动有效的抗生素治疗以防止耐药菌株传播至关重要。对于资源有限的环境而言，开发低成本、快速且可靠的耐多药结核病检测方法非常必要。
我们报告了使用基于内部质粒的定量聚合酶链反应-高分辨率熔解曲线分析（qPCR-HRM）来检测摩洛哥患者临床分离株中与利福平耐药结核分枝杆菌（MTB）相关的突变。五个含有主要突变（S531L、S531W、H526Y和D516V）以及利福平耐药决定区（RRDR）野生型序列的重组质粒已用作对照，以筛选45株利福平耐药和22株利福平敏感的MTB分离株。
与利福平药敏试验（DST）相比，qPCR-HRM分析的灵敏度和特异性分别为88.8%和100%。qPCR-HRM和DNA测序结果的一致性为100%。
我们的qPCR-HRM检测方法是一种灵敏、准确且经济高效的检测方法，可用于结核病参考实验室中基于突变的耐药性高通量筛选。
