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）：在资源有限的设置中，HIV病毒负载测量是持续需求的领域。分支DNA（BDNA）是一种基于多层溶液相探针结构的非PCR核酸方法，是满足资源有限设置中对HIV病毒载荷测量需求的有前途的方法。在参考实验室仪器中，分支的DNA化学支持在1 ml样品中少于75份病毒RNA的检测，而无需靶向扩增。分支DNA固有地抵抗样品污染，不需要热循环或极端温度，并且与封闭的cartridge-format测定仪器兼容。该阶段I项目着重于适合用于资源有限设置的墨盒形式BDNA系统开发的特定技术障碍：检测结合的BDNA探针杂交病毒RNA以确定测定结果。尽管标准BDNA化学发光检测在传统的临床实验室中产生了良好的结果，但该方法对旨在在紧密控制环境之外使用的BDNA系统提出了稳定性问题。为了克服这一挑战，提出了一种用于检测结合的bDNA探针杂交病毒RNA的新方法：在受振荡场的材料中非线性超级磁效应的跨调节峰检测。间调节峰检测是一种最近开发的磁性检测方法，它具有更良好的巨型磁势稳定方法的鲁棒性，但在检测空间分布的探针方面具有优势。最近已经证明了该方法的免疫测定方法，但据我们所知，此处提出的工作是首次将互化峰检测作为溶液 - 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BDNA测定敏感性，较少10个BDNA复合的病毒RNA分子在BDNA优化的多孔结构中，并且至少为4个数量级的测定动态范围。该项目工作包括基准级原型检测器构建以及广泛的实验，建模和分析。达到这些里程碑将证明可行性间调节峰检测是最终目标的组成部分，这是一个紧凑的，低维护的，电池供电的，由FDA批准的系统，该系统以两个小时的周转时间分析Fingerstick样品。拟议的研究是开创性的，探索了BDNA信号扩增与互育峰值检测的新型组合。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；它具有很高的价值，解决了部署诊断设备的重要技术障碍，并有可能在全球资源有限的环境中促进艾滋病毒患者人群的医疗水平的显着改善。该项目的中心假设是，在多孔结构内结合的bDNA复合物的间调节峰检测支持病毒RNA测量，并且限制的检测限量为每毫升或更高的5,000份拷贝，在动态范围的上限至少500,000份，每毫升至少500,000份，总测定持续时间小于120分钟。公共卫生相关性：由复杂的血液测试确定的血浆病毒载量表明患者患者的生病程度以及他或她对治疗的反应程度。常规的病毒负荷测量对于照顾艾滋病毒/艾滋病患者很重要，但是这些测试目前并不适合所有人。该项目探讨了使病毒负荷测量值更便宜的新方法，并为远离精致医疗机构的医生和患者更容易获得。