Basic Research for Diagnostics and Surveillance in Lower Resource Environments

浅层资源环境诊断与监测的基础研究 应用分子进化基础 史蒂文·A·本纳 摘要 我们将通过基础研究向NIAID和CDC社区提供对 天然DNA和RNA(统称为XNA)的配对、错配和酶学 公理“A与T配对，G与C配对”。这些实验旨在了解： (A)为什么在超过20-30个靶点的情况下，稳健的多重聚合酶链式反应(MPCR)似乎不可能用于临床。 (B)为什么采用传统的权宜之计(包括仔细的引子和探针设计、内部嵌套和外部嵌套 标记)无法有力地支持超过约30个目标的多路传输。 (C)为什么这些不合格不能在不同的样本中重现。 (D)为什么当增加第n个目标时，传统的以n个目标为目标的多路复用器经常崩溃。这 当出现新的病原体(如2019-nCoV)时，防止诊断人员简单地添加新的 以现有的mPCR试剂盒为目标，从而满足紧急需要。 (E)为什么随着多路复用水平的提高，制造规格变得越来越苛刻。 这些问题将21世纪的诊断学局限于20世纪的两种设计和监管范式。 (I)单链分子诊断的“先猜后测”模式，这需要医生猜测 哪种病原体可能与患者不适有关，根据这一点，开出一项约150美元的单基因测试 猜测，并重新开出进一步测试的处方，直到猜测被证明是正确的。 (2)“僵化--多路--小组”模式。在这里，分析被捆绑到适合于 特定样本和症状集；失败(D)防止该多重物因新出现的疾病而改变。 通过发展天然和非天然DNA的科学(包括人工扩增的基因 信息系统、宙斯盾和自我避免分子识别系统(SAMRS)，该项目将提供 对研究人员、制造商和FDA的科学来说，以满足21世纪NIAID的使命。我们会： 任务1.完成热力学规则和酶规则，以便将SAMRS最佳地放置在针对两个DNA的引物中 和RNA。规则将通过将使用这些规则做出的预测与实验进行比较来衡量。 任务2.通过深度测序来衡量mPCR故障(A)至(E)。 任务3.衡量Aegis和SAMRS如何减轻或消除故障(A)至(E)。 任务4.明确RNA靶标出现的失败模式。由于RNA具有折叠选项而不是 这些模式可用于DNA，可能尤其对核酸创新具有抵抗性。 任务5.建立宙斯盾-SAMRS mPCR统计知识体系，特别是关于“附加项目”， 定量放大和制造公差。这将有助于摆脱“猜想-然后- 测试“和”不灵活的多路复用板“范例，降低成本，支持FDA的监管程序， 以及更好地管理流行病。 1

Basic Research to Diagnostics and Surveillance in Lower Resource Environments Foundation for Applied Molecular Evolution Steven A. Benner ABSTRACT We will deliver to the NIAID and CDC communities, through basic research, a scientific understanding of pairing, mispairing, and enzymology of natural DNA and RNA (collectively xNA) that goes deeper than the axiom that "A pairs with T, and G pairs with C". The experiments are designed to learn: (a) Why robust multiplexed PCR (mPCR) for clinical use seems impossible with more than 20-30 targets. (b) Why conventional expedients (including careful primer and probe design, internal nesting, and external tagging) fail to robustly support multiplexing beyond ~30 targets. (c) Why those failures are not reproducible from sample to sample. (d) Why conventional multiplexes targeting n targets often collapse when an n+1th target is added. This prevents, when a new pathogen emerges (as for 2019-nCoV), a diagnostics maker from simply adding a new target to an existing mPCR kit, thereby meeting the emergency need. (e) Why manufacturing specs become increasingly more demanding as the level of multiplexing increases. These problems restrain 21st century diagnostics to two 20th century design and regulatory paradigms. (i) A "guess-then-test" paradigm for singleplexed molecular diagnosis, which requires physician to guess which pathogen might be associated with patient malaise, prescribe a ~$150 singleplexed test based on that guess, and re-prescribe further tests until a guess proves correct. (ii) The "inflexible-multiplexed-panel" paradigm. Here, assays are bundled into a multiplex appropriate for a specific sample and symptom set; failure (d) prevents that multiplex from changing for emerging diseases. By developing the science of both natural and unnatural DNA (including artificially expanded genetic information systems, AEGIS, and self avoiding molecular recognition systems, SAMRS), this project will deliver to researchers, manufacturers, and the FDA science to meet the 21st century NIAID mission. We will: Task 1. Complete thermodynamic and enzyme rules to place SAMRS optimally in primers that target both DNA and RNA. Rules will be metricked by comparing predictions made with these rules to experiments. Task 2. Metric, by deep sequencing, mPCR failures (a) through (e). Task 3. Metric how AEGIS and SAMRS mitigate or eliminate failures (a) through (e). Task 4. Identify failure modes that arise with RNA targets specifically. Since RNA has folding options not available to DNA, these modes may be especially resistant to nucleic acid innovations. Task 5. Build a body of statistical knowledge for AEGIS-SAMRS mPCR, especially with respect to "add-ons", quantitative amplification, and manufacturing tolerances. This will help move away from "guess-then- test" and "inflexible-multiplexed-panel" paradigms, lowing cost, supporting FDA regulatory processes, and better managing pandemics. 1