Real time PCR quantification of viable Mycobacterium tuberculosis from sputum samples treated with propidium monoazide
Real time PCR quantification of viable Mycobacterium tuberculosis from sputum samples treated with propidium monoazide
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DOI：
10.1016/j.tube.2014.04.008
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发表时间：
2014-07-01
期刊：
影响因子：
3.2
通讯作者：
Santos, Diogenes Santiago
中科院分区：
文献类型：
--
作者：
de Assuncao, Thiago Milech;Batista, Eraldo L., Jr.;Santos, Diogenes Santiago
如今，结核病的诊断方法很容易延误诊断，增加假阴性结果，并且对许多形式的少杆菌疾病不敏感。本研究的目的是对少杆菌结核病实施基于核酸的定量诊断测试，从而能够通过定量实时 PCR (qRT-PCR) 对活结核分枝杆菌进行鉴定和定量。选择单拷贝 inhA-mabA 基因的基因间区域作为设计与荧光团缀合的引物和探针的目标区域。目标区域侧翼的合成 DNA 的构建作为核酸绝对定量的标准。使用嵌入染料单叠氮化丙啶，我们能够区分结核分枝杆菌的活细胞和死细胞。仅与涂片阳性痰样本相比，诊断方法显示出广泛的敏感性（96.1%），ROC 分析表明，我们的方法表现良好，与结核分枝杆菌菌落形成单位计数相比，特异性为 84.6%，敏感性为 84.6%。 (C) 2014 Elsevier Ltd. 保留所有权利。
Diagnostic methods of TB, nowadays, are prone to delay in diagnosis, increased false negative results and are not sensitive to many forms of paucibacillary disease. The aims of this study were to implement a quantitative nucleic acid-based diagnostic test for paucibacillary tuberculosis, enabling the identification and quantification of viable Mycobacterium tuberculosis bacilli by quantitative Real-Time PCR (qRT-PCR). The intergenic region of the single-copy inhA-mabA gene was chosen as the target region for design of primers and probes conjugated with fluorophores. The construction of synthetic DNA flanking the target region served as standards for absolute quantification of nucleic acids. Using the intercaling dye, propidium monoazide, we were able to discriminate between viable and dead cells of M. tuberculosis. The diagnosis method showed a broad sensitivity (96.1%) when only compared to samples of smear-positive sputum and ROC analyses shows that our approach performed well and yielded a specificity of 84.6% and a sensitivity of 84.6% when compared to M. tuberculosis colony-forming units counting. (C) 2014 Elsevier Ltd. All rights reserved.