Intermodulation Peak Detection of Branched DNA for Compact-Apparatus Viral Load M

紧凑型装置病毒载量 M 的分支 DNA 互调峰检测

• 批准号: 7622727
• 负责人: Daniel James Laser
• 金额: $ 29.99万
• 依托单位: WAVE 80 BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7622727
• 关键词: AIDS Vaccines; AIDS/HIV problem; ATP8A2 gene; Academy; Acquired Immunodeficiency Syndrome; Address; Amplifiers; Antigens; Area; Autoimmune Diseases; Binding; Binding Sites; Biochemical; Biological Assay; Biological Markers; Biotin; Blood Tests; Body Fluids; Branched DNA Signal Amplification Assay; Calibration; Caring; Chemistry; Clinic; Clinical; Clinical Research; Clinical Trials; Communicable Diseases; Complex; Computer Systems Development; Consult; Controlled Environment; Coupling; DNA; DNA Probes; Data; Detection; Development; Devices; Diagnostic; Diamond; Disease Management; Engineering; Environmental Engineering technology; Equilibrium; Equipment and Supplies; FDA approved; Failure; Family; Foundations; Frequencies; Funding; Generations; Goals; HIV; Head; Health; Hour; Human; Human body; Immunoassay; In Vitro; International AIDS; Knowledge; Label; Laboratories; Lasers; Licensing; Life; Magnetism; Maintenance; Malaria; Malignant Neoplasms; Manufacturer Name; Maps; Measurement; Mechanics; Medical; Methods; Modeling; Natural Sciences; Noise; Nucleic Acids; Optics; Outcome; Patients; Performance; Phase; Plasma; Preparation; Principal Investigator; Process; Production; Proteins; Protocols documentation; Province; RNA; RNA Binding; Reading; Reagent; Reporter; Research; Research Personnel; Resistance; Resources; Running; Sampling; Science; Signal Transduction; Small Business Innovation Research Grant; Solid; Solutions; Streptavidin; Structure; Surface; System; Taiwan; Technology; Temperature; Testing; Therapeutic; Time; Viral Load result; Viral load measurement; Washington; Work; base; design; detector; experience; experimental analysis; extreme temperature; information processing; instrument; instrumentation; mathematical model; meetings; member; novel; patient population; product development; programs; prototype; public health relevance; research and development; stability testing; superparamagnetic beads; tool; viral RNA; voltage

DESCRIPTION (provided by applicant): HIV viral load measurement in resource-limited settings is an area of persistent need. Branched DNA (bDNA), a non-PCR nucleic acid method based on multitiered solution-phase probe structures, is a promising approach to meeting the need for HIV viral load measurement in resource-limited settings. On reference lab instruments, the branched DNA chemistry supports detection of as few as 75 copies of viral RNA in a 1 mL sample without target amplification. Branched DNA is inherently resistant to sample contamination, requires no thermal cycling or extreme temperatures, and is compatible with enclosed-cartridge-format assay instruments. This Phase I project focuses on a specific technical barrier to the development of cartridge-format bDNA systems suitable for use in resource-limited settings: detecting bound, bDNA probe-hybridized viral RNA to determine the assay outcome. While standard bDNA chemiluminescence detection yields excellent results in traditional clinical laboratories, this method presents stability concerns for bDNA systems intended for use outside of tightly controlled environments. To overcome this challenge, a novel method for detecting bound bDNA probe-hybridized viral RNA is proposed: intermodulation peak detection of nonlinear superparamagnetic effects in materials subjected to oscillating fields. Intermodulation peak detection is a recently developed magnetic detection method which shares the robustness of more well established giant magnetoresistance methods, but affords advantages in terms of detecting spatially distributed probes. This method has recently been demonstrated for immunoassays, but the work proposed here is (to our knowledge) the first use of intermodulation peak detection as a tool for solution-phase nucleic acid assays. Preliminary studies indicate that intermodulation peak detection methods can support bDNA assay sensitivities of as few as 10 bDNA-complexed viral RNA molecules within a bDNA-optimized porous structure and an assay dynamic range of at least four orders of magnitude. The project work encompasses bench-level prototype detector construction and extensive experimentation, modeling, and analysis. Reaching these milestones will demonstrate feasibility intermodulation peak detection as a component of the ultimate goal, a compact, low-maintenance, battery-powered, FDA-approved system which analyzes fingerstick samples with a two-hour turnaround time. The proposed research is pioneering, exploring a novel combination of bDNA signal amplification with intermodulation peak detection; it is rigorous, with exhaustive calibration protocols and careful mapping of relevant parameter spaces; and it is of high value, addressing a prominent technical barrier to the deployment of diagnostic devices with the potential to facilitate significant improvement in the level of care afforded HIV patient populations in resource-limited settings across the globe. The central hypothesis of this project is that intermodulation peak detection of bDNA complexes bound within a porous structure supports viral RNA measurement with a limit of detection of 5,000 copies per mL or better, upper bound of dynamic range at least 500,000 copies per mL, and total assay duration less than 120 minutes. PUBLIC HEALTH RELEVANCE: Plasma viral load, determined by complex blood tests, is an indication of how sick an HIV/AIDS patient is and how well he or she is responding to treatment. Regular viral load measurement is important in caring for HIV/AIDS patients, but these tests are currently not available for everyone. This project explores new ways of making viral load measurements less expensive and more readily available to doctors and patients who live far from sophisticated medical facilities.

描述（由申请人提供）：在资源有限的环境中测量HIV病毒载量是一个持续需要的领域。分支DNA（bDNA），一种基于多层液相探针结构的非PCR核酸方法，是一种很有前途的方法，可以满足在资源有限的环境中进行HIV病毒载量测量的需要。在参考实验室仪器上，分支DNA化学支持在没有靶扩增的情况下检测1 mL样品中的病毒RNA的低至75个拷贝。分支DNA具有固有的抗样品污染性，不需要热循环或极端温度，并且与双筒式分析仪器兼容。该I期项目的重点是开发适用于资源有限环境的嵌入式bDNA系统的特定技术障碍：检测结合的bDNA探针杂交病毒RNA以确定检测结果。虽然标准的bDNA化学发光检测在传统的临床实验室中产生了优异的结果，但该方法对用于严格控制环境之外的bDNA系统存在稳定性问题。为了克服这一挑战，提出了一种新的方法用于检测结合bDNA探针杂交病毒RNA：互调峰检测的非线性超顺磁效应的材料进行振荡场。互调峰值检测是最近开发的磁检测方法，其具有更完善的巨磁阻方法的鲁棒性，但在检测空间分布的探针方面提供优势。这种方法最近已被证明用于免疫测定，但这里提出的工作是（据我们所知）首次使用互调峰检测作为液相核酸测定的工具。初步研究表明，互调峰检测方法可以支持在bDNA优化的多孔结构内的少至10个bDNA复合的病毒RNA分子的bDNA测定灵敏度和至少四个数量级的测定动态范围。该项目的工作包括台架级原型探测器的建设和广泛的实验，建模和分析。达到这些里程碑将证明互调峰值检测作为最终目标的一个组成部分的可行性，最终目标是一个紧凑，低维护，电池供电，FDA批准的系统，分析手指针刺样本，两小时的周转时间。拟议的研究是开创性的，探索了bDNA信号放大与互调峰检测的新组合;它是严格的，具有详尽的校准协议和相关参数空间的仔细映射;并且它具有很高的价值，解决了部署诊断设备的一个突出技术障碍，有可能促进在资源方面显著提高艾滋病毒患者群体的护理水平，地球仪的有限设置。本项目的中心假设是结合在多孔结构内的bDNA复合物的互调峰检测支持病毒RNA测量，检测限为5，000拷贝/mL或更高，动态范围上限至少为500，000拷贝/mL，总测定持续时间小于120分钟。公共卫生相关性：血浆病毒载量，通过复杂的血液测试确定，是艾滋病毒/艾滋病患者病情如何以及他或她对治疗反应如何的指标。定期测量病毒载量对于照顾艾滋病毒/艾滋病患者很重要，但这些测试目前并不适用于所有人。该项目探索了新的方法，使病毒载量测量更便宜，更容易为医生和远离先进医疗设施的患者提供。