Amplified detection of viral RNA using catalytic DNA logic circuits

使用催化 DNA 逻辑电路放大检测病毒 RNA

基本信息

批准号：
8806318
负责人：
STEVEN W GRAVES
金额：
$ 18.88万
依托单位：
UNIVERSITY OF NEW MEXICO
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-12-01 至 2016-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8806318
关键词：
Address Air Area Biological Biological Assay Biomedical Research Catalytic DNA Clinical Clinical Protocols Communities Coronavirus Coupling DNA Data Dengue Detection Development Devices Diagnosis Disease Disease Outbreaks Epidemic Equipment Escherichia coli Flaviviridae Flavivirus Fluorescence Fright Future Genomic DNA Goals Health Health Personnel Healthcare Human Immunoglobulins In Vitro Incidence Infection Influenza Influenza A Virus, H5N1 Subtype Kinetics Laboratories Lateral Liquid substance Logic Measurement Medical Medicine Middle East Respiratory Syndrome Coronavirus Military Personnel Modeling Molecular Molecular Computations Morbidity - disease rate Nanotechnology Nature Noise Outcome Patients Performance Preparation Property Protocols documentation RNA RNA Viruses RNA amplification Reaction Reporting Research Resource Allocation Reverse Transcriptase Polymerase Chain Reaction Sampling Science Serotyping Serum Severe Acute Respiratory Syndrome Shiga Toxin Signal Transduction St. Louis Encephalitis Virus Technical Expertise Techniques Technology Testing Training Travel Tube United States Viral Viral Genome Viral Load result Virulent Virus Virus Diseases West Nile virus Work base climate change combat design feeding flexibility improved influenzavirus interest mortality operation pandemic disease pathogen pathogenic bacteria point of care public health relevance response success theranostics tool vector mosquito viral RNA viral detection

项目摘要

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.

描述（由适用提供）：我们建议开发基于等温的，基于分子逻辑的测定，以扩大病毒RNA的检测，以创建一个强大的，灵活的测定框架，以用于临床样品中的病毒检测。该框架的优点将包括感知多个目标并将信息组合到单个读数中以减少误报的能力，将等温目标放大到可检测的信号，直接在现场应用中的直接操作以及启用快速设计的能力，并启用简单的设计反对新兴病原体菌株的重新定位。这些是实用病毒检测测定法的非常理想的特性。简单，温病毒检测测定法的发展具有很大的医学意义，因为病毒爆发引起的疾病代表了全球的重大医疗负担。例如，有影响力的人既负责季节性暴发和高度致病的菌株，例如H5N1。这些病毒株不断发展，这使得可以快速对新的人类适应性菌株进行迅速重新定位测定框架至关重要。由于气候变化，在美国对实用，准确的病毒检测测定法进一步驱动了热带病毒（例如Flaviviridae）。我们将通过整合催化分子逻辑电路来开发等温的，基于逻辑的病毒检测测定法，并通过等温RNA扩增在体外成功证明了这一点。我们提出的平台将使用诸如滚动圆扩增等技术在医学相关的病毒滴度上感知多个目标，并将放大信号馈入多输入的分子逻辑回路以产生集成的响应，我们将使用荧光测量结果检测到。该平台的成功开发将在针对血清中RNA低聚物的生物学现实模型测定中得到证明。然后，这些测定将用于检测和血清型风扇在临床样本中的感染，并根据反应的动力学来得出病毒载荷的估计。这些目标将需要开发具有较低背景响应和高信噪比的分子逻辑设备，并且可以抵抗生物液体中降解的较高的信噪比，增强了纯化方案，用于构建高质量的分子逻辑设备，以及适合临床样品的合适样品制备方案。如果成功，我们的测定将达到医学上相关的检测限制并且将直接执行，并且易于针对新的病毒株进行重新定位。除了拟议的病毒检测研究的直接应用外，我们的评估框架还将广泛适用于其他生物医学研究领域。例如，使用合适的样品制备方案，我们的装置可以针对基因组DNA，从而可以检测病原细菌，例如含志毒素的大肠杆菌。此外，我们评估的分子逻辑成分将引起DNA纳米技术社区的关注，并在自主疗法中应用。拟议的工作将对科学和医学都有宽泛的一般利益。