Improved HIV Assays by Combining Four Innovations in Nucleic Acid Chemistry

描述（由申请人提供）：2010年，Benner小组宣布开发了四项与检测复杂生物样本中人类免疫缺陷病毒（HIV）的工具相关的创新：（a）支持“六核苷酸PCR”的人工扩增遗传信息系统（AEGIS），允许独立扩增少量HIV RNA，而不受环境中其他DNA的干扰。(b)一种自避免分子识别系统（SAMRS），支持DNA探测、引发和多重PCR扩增中基本上无限的多重化。(c)将标准DNA转化为含AEGIS的DNA的程序，支持下游正交捕获，使DNA靶向测定具有灵活性和适应性，可能允许将新靶标添加到多重测定试剂盒中，而不需要对试剂盒中已靶向的部分进行返工。(d)可逆终止子，作为三磷酸，假设允许检测和相对定量变异HIV序列。 我们假设，通过结合这些创新，我们可以改进HIV诊断工具，扩大其能力，在更复杂的生物环境中检测更少的病毒体，具有更大的动态范围和更高的亚型特异性，以及更大的多重性。此外，这些技术应该提供灵活性;这应该是可能的， 快速增加检测新变种、并发感染因子甚至识别 在诊断HIV感染的过程中，在HIV基因组的特定位点上发现以前未知的变异。 为了验证这一假设，我们将进行一系列分阶段的检测开发，将这些创新中的每一个系列添加到HIV靶序列检测中日益具有挑战性的问题中，从相对简单环境中单个HIV靶标的单重检测开始，随着我们降低靶分子的量，增加多路复用水平，并使环境更加复杂，增加创新。在每个阶段，我们将驱动系统失败，并记录系统失败的参数（灵敏度，复杂度，多路复用级别）。这些定义了一个“参数空间”，提供了一个衡量进展的指标。该项目还将提供可交付的引物、探针和检测捕获珠试剂盒，供有兴趣进行基准测试或使用它们的艾滋病毒研究人员使用。 虽然Benner实验室的技术支持现在广泛用于测量HIV病毒载量的分支DNA（bDNA）3.0工具，但这是Benner实验室首次为艾滋病研究寻求资金。因此，这项工作的另一个目标将是允许Benner实验室的创新更广泛地用于解决NIAID的许多HIV相关问题。这将有助于NIAID帮助实现国家艾滋病毒/艾滋病战略制定的目标，即到2015年将美国对艾滋病毒状况的认识从79%提高到90%。

DESCRIPTION (provided by applicant): In 2010, the Benner group announced the development of four innovations relevant to tools to detect human immunodeficiency virus (HIV) in complex biological samples: (a) An artificially expanded genetic information systems (AEGIS) that supports "six nucleotide PCR", allowing independent amplification of small amounts of HIV RNA without interference from other DNA in the environment. (b) A self-avoiding molecular recognition system (SAMRS) that supports essentially unlimited multiplexing in DNA probing, priming, and multiplexed PCR amplification. (c) Procedures that convert standard DNA into AEGIS-containing DNA, supporting downstream orthogonal capture that allows DNA-targeted assays to be flexible and adaptive, possibly allowing new targets to be added to a multiplexed assay kit without demanding a reworking of the parts of that kit already targeted. (d) Reversible terminators that, as triphosphates, are hypothesized to allow detection and relative quantitation of variant HIV sequences. We hypothesize that by combining these innovations, we can improve HIV diagnostics tools, expanding their power to detect fewer virions in more complex biological environments with greater dynamic range and greater subtype specificity, together greater multiplexing. Further, these technologies should deliver flexibility; it should be possible to rapidly add capabilities to detect new variants, co-incident infectious agents, or even identify previously unknown variants at specific sites in the HIV genome in the course of diagnosing HIV infections. To test this hypothesis, we will perform a staged series of assay development, adding each of these innovations in series to increasingly challenging problems in the detection of HIV target sequences, starting with singleplexed detection of single HIV targets in relatively simple environments, adding innovations as we lower the amount of target molecules, increase the level of multiplexing, and make the environment more complex. At each stage, we will drive the system to fail, and note the parameters (sensitivity, complexity, multiplexing level) at which the system fails. These define a "parameter space" which provides a metric for progress. This project will also make available as deliverables kits of primers, probes, and detection capture beads, to be provided HIV researchers interested in benchmarking or using them. Although technology from the Benner laboratory stands behind the branched DNA (bDNA) 3.0 tool now widely used to measure HIV viral load, this is the first time that the Benner laboratory has sought funding for AIDS research. Thus, a further goal of this work will be to allow innovations from the Benner laboratory to be more widely used to solve the many HIV-related problems at the NIAID. This will help the NIAID help meet the goal established by the National HIV/AIDS Strategy of increasing the awareness of HIV status from 79% to 90% by 2015 in the US.