Bengtson Hillary N, Homolka Susanne, Niemann Stefan, Reis Ana Júlia, da Silva Pedro Eduardo, Gerasimova Yulia V, Kolpashchikov Dmitry M, Rohde Kyle H
Division of Immunity and Pathogenesis, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, USA.
Molecular and Experimental Mycobacteriology, Research Center Borstel, Borstel, Germany.
Biosens Bioelectron. 2017 Aug 15;94:176-183. doi: 10.1016/j.bios.2017.02.051. Epub 2017 Mar 1.
Current diagnostic tools for Mycobacterium tuberculosis (Mtb) have many disadvantages including low sensitivity, slow turnaround times, or high cost. Accurate, easy to use, and inexpensive point of care molecular diagnostic tests are urgently needed for the analysis of multidrug resistant (MDR) and extensively drug resistant (XDR) Mtb strains that emerge globally as a public health threat. In this study, we established proof-of-concept for a novel diagnostic platform (TB-DzT) for Mtb detection and the identification of drug resistant mutants using binary deoxyribozyme sensors (BiDz). TB-DzT combines a multiplex PCR with single nucleotide polymorphism (SNP) detection using highly selective BiDz sensors targeting loci associated with species typing and resistance to rifampin, isoniazid and fluoroquinolone antibiotics. Using the TB-DzT assay, we demonstrated accurate detection of Mtb and 5 mutations associated with resistance to three anti-TB drugs in clinical isolates. The assay also enables detection of a minority population of drug resistant Mtb, a clinically relevant scenario referred to as heteroresistance. Additionally, we show that TB-DzT can detect the presence of unknown mutations at target loci using combinatorial BiDz sensors. This diagnostic platform provides the foundation for the development of cost-effective, accurate and sensitive alternatives for molecular diagnostics of MDR- and XDR-TB.
目前用于结核分枝杆菌（Mtb）的诊断工具存在许多缺点，包括灵敏度低、周转时间长或成本高。对于作为公共卫生威胁在全球出现的耐多药（MDR）和广泛耐药（XDR）Mtb菌株的分析，迫切需要准确、易用且廉价的即时分子诊断测试。在本研究中，我们建立了一种用于Mtb检测和使用二元脱氧核酶传感器（BiDz）鉴定耐药突变体的新型诊断平台（TB-DzT）的概念验证。TB-DzT将多重聚合酶链反应（PCR）与单核苷酸多态性（SNP）检测相结合，使用针对与菌种分型以及对利福平、异烟肼和氟喹诺酮类抗生素耐药相关位点的高度选择性BiDz传感器。使用TB-DzT检测方法，我们证明了在临床分离株中能够准确检测Mtb以及与对三种抗结核药物耐药相关的5种突变。该检测方法还能够检测耐药Mtb的少数群体，即临床上称为异质性耐药的相关情况。此外，我们表明TB-DzT可以使用组合BiDz传感器检测目标位点未知突变的存在。该诊断平台为开发用于MDR-TB和XDR-TB分子诊断的经济高效、准确且灵敏的替代方法奠定了基础。
