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

描述(由申请人提供)：在资源有限的环境中测量艾滋病毒病毒载量是一个长期需要的领域。分支DNA(BDNA)是一种基于多层溶液相探针结构的非聚合酶链式反应核酸方法，是一种在资源有限的环境下满足HIV病毒载量测量需求的有前途的方法。在参考实验室仪器上，分支DNA化学支持在没有目标扩增的情况下在1毫升样本中检测到低至75个拷贝的病毒RNA。分支DNA固有地抵抗样本污染，不需要热循环或极端温度，并且与封闭式卡盒格式的分析仪器兼容。该第一阶段项目侧重于开发适用于资源有限环境的盒式BDNA系统的一个特定技术障碍：检测结合的BDNA探针杂交的病毒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出色的结果，但这种方法对用于严格控制环境之外的BDNA系统的稳定性提出了担忧。为了克服这一挑战，提出了一种检测结合BDNA探针杂化病毒RNA的新方法：振荡场作用下材料中非线性超顺磁性效应的互调峰值检测。互调峰值检测是最近发展起来的一种磁检测方法，它具有更成熟的巨磁电阻方法的稳健性，但在检测空间分布的探针方面具有优势。这种方法最近已经被证明用于免疫分析，但据我们所知，这里提出的工作是(据我们所知)第一次使用互调峰检测作为溶液相核酸分析的工具。初步研究表明，互调峰检测方法可以支持在BDNA优化的多孔结构中对10个BDNA复杂的病毒RNA分子进行BDNA检测的灵敏度，以及至少四个数量级的检测动态范围。该项目的工作包括实验室级探测器原型的建造以及广泛的实验、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件的可行性，这是一个紧凑、低维护、电池供电的FDA批准的系统，可以在两个小时的周转时间内分析手指样本。这项拟议的研究具有开创性，探索了BDNA信号放大与互调峰值检测的新组合；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；它具有很高的价值，解决了部署诊断设备的突出技术障碍，有可能促进在全球资源有限的环境中为艾滋病毒患者提供的护理水平显著提高。该项目的中心假设是，结合在多孔结构中的BDNA复合体的互调峰检测支持病毒RNA测量，检测的极限为每毫升5,000个拷贝或更好，动态范围的上限至少为每毫升500,000个拷贝，总分析持续时间小于120分钟。与公共卫生相关：血浆病毒载量由复杂的血液测试确定，是艾滋病毒/艾滋病患者病情严重程度和对治疗反应良好的指标。定期病毒载量测量对于护理艾滋病毒/艾滋病患者很重要，但目前并不是每个人都可以进行这些测试。该项目探索了使病毒载量测量更便宜、更容易获得的新方法，供居住在远离先进医疗设施的医生和患者使用。

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.