Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
J Appl Microbiol. 2012 Oct;113(4):856-62. doi: 10.1111/j.1365-2672.2012.05379.x. Epub 2012 Aug 21.
This study was carried out to evaluate high-resolution melting (HRM) curve analysis assay for detection of mutations in three drug resistance-associated genes of Mycobacterium tuberculosis.
Clinical isolates of Myco. tuberculosis phenotypically resistant to rifampicin (n = 29), isoniazid (n = 35) and streptomycin (n = 34) were analysed for mutations in rpoB, katG and rpsL genes, respectively, by HRM curve analysis and DNA sequencing. HRM curve assay resulted in 11 clearly distinguishable melt curves denoting eight types of mutations responsible for drug resistance. For the three drugs, respectively, the sensitivity of HRM curve assay was found to be 93·1, 80 and 61·8% compared to the phenotypic resistance patterns, and 93·1, 93·3 and 100% in comparison with the DNA sequencing.
The sensitivity and specificity of HRM curve assay was found to be comparable to DNA sequencing. The assay offers the advantage of high throughput, single step, rapid work flow and cost effectiveness and can be utilized as a rapid screening method for detection of drug-resistant tuberculosis.
HRM curve assay may prove to be an important tool for the development of rapid molecular diagnostic assays for detection of mutation-based drug resistance.
本研究旨在评估高分辨率熔解（HRM）曲线分析检测结核分枝杆菌三种耐药相关基因突变的方法。
对表型耐利福平（n=29）、异烟肼（n=35）和链霉素（n=34）的临床分离株进行 rpoB、katG 和 rpsL 基因的 HRM 曲线分析和 DNA 测序，以检测突变。HRM 曲线分析产生了 11 个明显可区分的熔解曲线，表明了导致耐药的 8 种类型的突变。对于这三种药物，HRM 曲线分析的敏感性分别为 93.1%、80%和 61.8%，与表型耐药模式相比，与 DNA 测序相比，敏感性分别为 93.1%、93.3%和 100%。
HRM 曲线分析的敏感性和特异性与 DNA 测序相当。该检测方法具有高通量、单步、快速工作流程和成本效益的优势，可作为检测耐药性结核的快速筛选方法。
HRM 曲线分析可能成为基于突变的耐药性快速分子诊断检测的重要工具。
