Paper-based HIV self test

摘要：目前对艾滋病毒的自我检测依赖于对病毒抗体的检测，因此不是 在感染的早期阶段很有用。这些化验方法也不适用于 可能有病毒反弹风险的抗逆转录病毒治疗者，因为先前存在抗HIV抗体 掩盖病毒载量的变化。相反，当前基于病毒RNA检测的艾滋病毒诊断 是敏感的，但太复杂和昂贵，不适合进行自我测试。要解决这个问题 迫切需要，我们将开发第一个无酶样本答案艾滋病毒病毒载量自我测试，在 其中手指体积的血液被被动处理，导致分离出的 HIV基因组RNA。RNA扩增策略将依赖于发夹级联反应(HCR)， 一种DNA纳米技术方法，在其他情况下具有已证明的单拷贝敏感性，在固着的 Drop实现。整个测试将在一种廉价的、一次性的、 纸质消耗品。在R61阶段，我们建议：(1)在我们已有的方法基础上构建 之前经过验证，优化了全血与纸基病毒的被动处理 裂解和RNA分离；(2)完全建立基于HCR的HIV基因组扩增 RNA；(3)将样品处理和RNA扩增整合为一种消耗品；以及(4) 从有艾滋病毒感染风险的患者那里获得关于检测设计和可用性的反馈，并开发 一个试验性的制造计划。在R33阶段，我们将(1)规模化生产试剂盒 适合中试测试的合同制造商数量，以及(2)完成化验测试 有1000名艾滋病毒感染风险患者的队列，从访问 罗切斯特大学医学中心的急诊科。

Abstract: Current self-tests for HIV rely on detection of antibodies to the virus, and thus are not useful during the early stages of infection. These assays are also not appropriate for patients on antiretroviral therapy who may be at risk for viral rebound, as pre-existing anti-HIV antibodies mask changes in viral load. Conversely, current HIV diagnostics based on detection of viral RNA are sensitive, but too complex and expensive to be appropriate for self-testing. To address this critical need, we will develop the first enzyme-free sample-to-answer HIV viral load self-test, in which a fingerstick volume of blood is passively processed leading to amplification of the isolated HIV genomic RNA. The RNA amplification strategy will rely on hairpin cascade reactions (HCR), a DNA nanotechnology approach with proven single-copy sensitivity in other contexts, in a sessile droplet implementation. The entire assay will be implemented in an inexpensive, disposable, paper-based consumable. In the R61 phase, we propose to: (1) building on methods we have previously validated, optimize passive processing of whole blood coupled with paper-based viral lysis and RNA isolation; (2) complete development of HCR-based amplification of HIV genomic RNA; (3) integrate sample processing and RNA amplification into a single consumable; and (4) obtain feedback on assay design and usability from patients at risk for HIV infection, and develop a pilot manufacturing plan. In the R33 phase, we will (1) scale production of the assay with contract manufacturers in quantities suitable for pilot testing, and (2) complete testing of the assay with a cohort of 1000 patients at-risk for HIV infection, recruited from patients visiting the Emergency Department at the University of Rochester Medical Center.