Amplified detection of viral RNA using catalytic DNA logic circuits

 描述（由申请方提供）：我们提议开发用于病毒RNA扩增检测的等温、基于分子逻辑的检测方法，以创建用于临床样本中病毒检测的强大、灵活的检测框架。该框架的优点将包括感测多个靶标并将信息联合收割机组合在单个读出中以减少假阳性的能力、等温扩增低靶标浓度为可检测信号的能力、用于现场应用的简单操作以及使得能够进行快速检测的简单设计。 重新定位新出现的病原体菌株。这些对于实际的病毒检测测定是高度期望的性质。开发简单的等温病毒检测方法具有重要的医学意义，因为病毒爆发引起的疾病代表了重大的全球医疗负担。例如，流感病毒是季节性爆发和高致病性毒株如H5N1的原因。这些病毒株不断进化，使得检测框架能够快速重新靶向新的人类适应株变得至关重要。由于气候变化，热带病毒如黄病毒科的发病率增加，进一步推动了美国对实用、准确的病毒检测测定的需求。我们将开发等温的，基于逻辑的病毒检测检测方法，通过整合催化分子逻辑电路，我们已经成功地在体外证明，与等温RNA扩增。我们提出的平台将使用滚环扩增等技术来检测医学相关病毒滴度的多个目标，并将放大的信号输入多输入分子逻辑电路以产生集成响应，我们将使用荧光测量进行检测。该平台的成功开发将在针对血清中RNA寡聚体的生物现实模型测定中得到证实。然后，将使用这些检测方法检测临床样本中的登革热感染并进行血清分型，并根据反应动力学得出病毒载量的估计值。这些目标将需要开发具有低背景响应和高信噪比的分子逻辑器件，其抵抗生物流体中的降解，用于构建高质量分子逻辑器件的增强的纯化方案，以及用于临床样品的合适的样品制备方案。如果成功，我们的检测将达到医学相关的检测限 并且操作简单，易于针对新的病毒株重新定位。除了病毒检测研究的直接应用外，我们的检测框架将广泛适用于生物医学研究的其他领域。例如，通过合适的样品制备方案，我们的设备可以靶向基因组DNA，从而能够检测致病菌，例如携带滋贺毒素的大肠杆菌。杆菌此外，我们的分析的分子逻辑组件将感兴趣的DNA纳米技术社区，在自主治疗诊断学的应用。因此，所提出的工作将对科学和医学都有广泛的普遍利益。

 DESCRIPTION (provided by applicant): We propose to develop isothermal, molecular logic-based assays for the amplified detection of viral RNA, to create a powerful, flexible assay framework for virus detection in clinical samples. The advantages of this framework will include the ability to sense multiple targets and combine the information in a single readout to reduce false positives, the ability to isothermally amplify low target concentrations into a detectable signal, straightforward operation for application in the field, and a simple design to enable rapid retargeting against emerging pathogen strains. These are highly desirable properties for a practical virus detection assay. The development of simple, isothermal virus detection assays has great medical significance, as the illnesses caused by viral outbreaks represent a significant global healthcare burden. For example, influenza viruses are responsible both for seasonal outbreaks and for highly pathogenic strains such as H5N1. These viral strains are continually evolving, making it essential that assay frameworks can be rapidly retargeted against new human-adapted strains. The need for practical, accurate virus detection assays in the United States is further driven by increased incidence of tropical viruses such as Flaviviridae, due to climate change. We will develop isothermal, logic-based virus detection assays by integrating catalytic molecular logic circuits, which we have successfully demonstrated in vitro, with isothermal RNA amplification. Our proposed platform will use techniques such as rolling circle amplification to sense multiple targets at medically relevant viral titers and feed the amplified signals into multi-input molecular logic circuits to produce an integrated response, which we will detect using fluorescence measurements. Successful development of this platform will be demonstrated in biologically realistic model assays against RNA oligomers in serum. The assays will then be used to detect and serotype dengue infections in the clinical samples and to derive estimates of the viral load based on the kinetics of the response. These goals will require the development of molecular logic devices with low background responses and high signal-to-noise ratios that resist degradation in biological fluids, enhanced purification protocols for the construction of high-quality molecular logic devices, and suitable sample preparation protocols for clinical samples. If successful, our assays will achieve medically relevant limits of detection and will be straightforward to perform and easy to retarget against new viral strains. Beyond the immediate applications of the proposed research for virus detection, our assay framework will be broadly applicable in other areas of biomedical research. For example, with suitable sample preparation protocols our devices could be targeted against genomic DNA, enabling the detection of pathogenic bacteria such as Shiga toxin-bearing E. coli. Furthermore, the molecular logic components of our assays will be of interest to the DNA nanotechnology community, with applications in autonomous theranostics. Thus the proposed work will have widespread general benefit both to science and to medicine.