Improved HIV Assays by Combining Four Innovations in Nucleic Acid Chemistry

描述（由申请人提供）：2010年，Benner小组宣布开发了与在复杂生物样品中检测人类免疫缺陷病毒（HIV）的工具相关的四项创新：(a)支持“六核苷酸PCR”的人工扩展遗传信息系统（AEGIS），允许独立扩增少量HIV RNA，而不受环境中其他DNA的干扰。(b)一个自我回避的分子识别系统（SAMRS），基本上支持DNA探测、引物和多重PCR扩增的无限复用。(c)将标准DNA转化为含有aegis的DNA的程序，支持下游正交捕获，使DNA靶向分析具有灵活性和适应性，可能允许将新靶标添加到多路分析试剂盒中，而无需对试剂盒中已经靶向的部分进行重新加工。(d)可逆终止体，作为三磷酸盐，假设可以检测和相对定量变异的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粒子，具有更大的动态范围和更大的亚型特异性，以及更大的多路复用。此外，这些技术应该提供灵活性；这应该是可能的

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.