DNA circuits for point-of-care diagnostics

描述(由申请人提供)：我们建议调整新颖的，熵驱动的DNA电路，这是最近才开发出来的，以满足医疗保健点诊断的迫切需求。通过与特定的mRNA等位基因杂交，亚稳态DNA电路将被激活，并将经历一系列链交换放大反应，要么是线性的，要么是指数的，最终产生一种脱氧核酶过氧化物酶，可以产生易于阅读的比色信号。我们将使用一种新的优化技术，顺序注射分析，以找到在各种样品和环境条件下提供稳健信噪比的反应条件。我们将应用优化的DNA电路检测来自阿富汗的痰标本中结核分枝杆菌对利福平耐药的RNA聚合酶等位基因。这些实验应该为开发一种用于耐药结核分枝杆菌感染的定点诊断的单管比色分析奠定基础。 公共卫生相关性：结合新的化验技术、新的优化方法和该领域的现实应用，对于推进公共卫生监测和维护的选择，其影响不可低估。自我相同的DNA电路可以很容易地重新设计，以检测其他mRNA和等位基因，因此也应该在第一世界的临床环境中找到巨大的用途。

DESCRIPTION (provided by applicant): We propose to adapt novel, entropy-driven DNA circuitry that has only recently been developed to pressing needs in point-of-care diagnostics. The metastable DNA circuits will be activated by hybridization to a specific mRNA allele, and will undergo a series of strand exchange amplification reactions, either linear or exponential, culminating in the production of a deoxyribozyme peroxidase that can generate an easy-to-read colorimetric signal. We will use a novel optimization technique, sequential injection analysis, to find reaction conditions that give robust signal-to-noise ratios under a variety of sample and environmental conditions. We will apply the optimized DNA circuitry to the detection of rifampicin resistant RNA polymerase alleles in M. tuberculosis in sputum samples obtained from Afghanistan. These experiments should set the stage for the development of a one-tube, colorimetric assay for point-of-care diagnosis of drug resistant M. tuberculosis infection. PUBLIC HEALTH RELEVANCE: The impact of marrying novel assay techniques, novel optimization methods, and realistic applications in the field cannot be underestimated for advancing options for public health monitoring and maintenance. The selfsame DNA circuitry can be readily redesigned for the detection of other mRNAs and alleles, and should therefore find great use in first world clinical settings, as well.