Self-Powered Sample Concentrating and CRISPR-based Biosensing for Moile HIV-1 RNA Detection

自力式样品浓缩和基于CRISPR的移动生物传感 HIV-1核糖核酸检测 摘要 艾滋病毒/艾滋病已成为影响全世界约3790万人的重大公共卫生问题。早期诊断 在血清转换窗口期发现急性HIV感染将有助于早期干预。在抗逆转录病毒期间 艾滋病病毒感染者的治疗(ART)，需要经常监测艾滋病毒病毒载量以确认治疗 有效性，并识别病毒反弹。HIV病毒载量测试，量化HIV病毒RNA(循环中的HIV 血浆中的病毒)是ART监测和急性HIV检测最准确和可靠的方法。 然而，目前标准的艾滋病毒病毒载量检测方法依赖于昂贵的设备和训练有素的 限制了其在集中式实验室和医院环境中的临床应用。 基于免疫分析的医疗点(POC)诊断技术，如OraQuick® 艾滋病毒自检(HIVST)对于检测急性艾滋病毒感染以及抗逆转录病毒治疗失败并不有效。作为一名 结果，由于缺乏一种简单、快速、负担得起的艾滋病毒RNA检测POC诊断工具，留下了许多 没有意识到自己病情的个人，阻碍了及时的抗逆转录病毒治疗。为了填补这一空白，我们建议 开发一种低成本(~5美元)、快速(&lt；35分钟)和敏感(&lt；1000份/毫升)、定期聚集的 用于HIV病毒载量检测的间隔短回文重复序列(CRISPR)生物传感平台 穿刺量(~50微米L)全血。在R61阶段(目标1-3)，我们将：i)高度发展和优化 用于新一代核酸分子的灵敏、特异的CRISPR生物传感技术 诊断，以及ii)设计和制造一次性“血液对答案”、CRISPR生物传感设备，该设备 集成了自助式血浆分离、病毒RNA浓缩和CRISPR生物传感检测。在 33阶段(目标4-5)，我们将系统地评估我们的集成CRISPR生物传感的性能 平台，并通过检测美国HIV临床样本严格验证其临床应用的可行性 和赞比亚。如果成功，这样一个简单、快速、负担得起的POC检测平台将使急性艾滋病毒 在家中进行诊断和病毒负载测试，并适用于艾滋病毒最多的资源有限的环境 很流行。

Self-Powered Sample Concentrating and CRISPR-based Biosensing for Mobile HIV-1 RNA Detection Abstract HIV/AIDS has become a major public health concern affecting ~37.9 million people worldwide. Early diagnosis of acute HIV infection during seroconversion window will facilitate early intervention. During antiretroviral treatment (ART) of HIV-infected patients, it requires frequent monitoring of HIV viral load to confirm treatment effectiveness, and to identify viral rebound. HIV viral load testing that quantifies HIV viral RNA (circulating HIV virus) in plasma is the most accurate and reliable approach for the ART monitoring and acute HIV detection. However, current standard HIV viral load testing methods rely on expensive equipment and well-trained personnel, limiting their clinical applications in centralized laboratories and hospital environments. Commercially available immunoassay-based point of care (POC) diagnostic technologies, such as OraQuick® HIV Self-Test (HIVST), are not effective to detect acute HIV infections, as well as ART failure. As a consequence, the lack of a simple, rapid, affordable, POC diagnostic tool for HIV RNA detection leaves many individuals unaware of their condition and impedes timely antiretroviral treatment. To fill this gap, we propose to develop a low-cost (~ $ 5), rapid (< 35 min), and sensitive (<1,000 copies/mL), clustered regularly interspaced short palindromic repeats (CRISPR) biosensing platform for HIV viral load testing using finger- prick volume (~50 µL) of whole blood. In the R61 phase (Aims 1-3), we will: i) develop and optimize highly sensitive and specific CRISPR biosensing technology for next-generation nucleic acid-based molecular diagnostics, and ii) design and fabricate a disposable "blood-to-answer", CRISPR biosensing device that integrates self-powered plasma separation, viral RNA enrichment, and CRISPR biosensing detection. In the Phase 33 (Aims 4-5), we will systematically evaluate the performance of our integrated CRISPR biosensing platform, and rigorously validate its feasibility for clinical application by testing HIV clinical samples in the US and Zambia. If successful, such a simple, rapid, affordable, POC detection platform will enable acute HIV diagnosis and viral load testing at home and be appropriate for resource-limited settings where HIV is most prevalent.