An integrated platform for blood collection, HIV viral RNA purification, and stab

描述(由申请人提供)：经济、后勤和物质障碍限制艾滋病毒病毒载量量化，艾滋病毒诊断的黄金标准。测试的成本很高(每次测试大约100美元)，而且所需的复杂设备将测试限制在配备训练有素的技术人员的实验室。此外，生物标志物病毒RNA的不稳定性质要求在抽血后6小时内进行检测，除非RNA能够稳定下来，因此限制了这一检测的地理范围。目前的临床点(POC)HIV检测依赖于抗体效价，但这种生物标志物仅限于早期、急性阶段的感染，导致许多假阴性。此外，保存血样(例如，干血点)的策略增加了工作流程中的额外步骤(例如，点样、干燥、洗脱)，导致增加了劳动力并增加了出错的可能性。我们提出了一种装置，它融合了Tasso Inc.在商业开发中的一种新的血液采集方法，以及UW-Madison开发的一种样品制备和稳定技术。通过一种集血液采集和样本制备于一体的简单易用的方法，该设备将缓解艾滋病毒诊断应用的许多障碍(时间、成本、训练有素的专家)。此外，一种新的样品稳定方法使该方法能够在任何数量的低资源环境中使用和运输，从而扩大了血液诊断方法的覆盖范围。为了实现这一项目，Tasso通过三项关键合作处于独特的地位：1)David Beebe教授是简单微流控设备的专家顾问，在4家不同的公司有创业经验；2)Frank Graziano博士是低资源环境下的诊断专家顾问，与乌干达的联合临床研究中心(JCRC)有特殊的联系；3)摩格里奇研究所，使Tasso能够直接访问现有的原型设施，并接触到可制造性设备设计方面的专家(包括TomoTreatment的创始人Thomas Mackie)。该提案包括三个目标。首先，Tasso用于血液采集的设备将通过使设备的非血液接触部分可重复使用来进行经济有限的部署。下一步，一步样品制备盒，即幻灯片，将进行优化，以从用Tasso设备采集的血液样本中提取病毒RNA。最后，基于蜡基的密封机制将被集成到载玻片中，以实现芯片上的逆转录，从而将病毒RNA稳定到DNA中，并密封以便于运输。在第一阶段结束时，一个集成的、优化的概念验证平台将能够采集血液、提取病毒RNA以及稳定和储存RNA。在第二阶段，这一原型将在顾问弗兰克·格拉齐亚诺博士的协助下在患者身上进行测试，格拉齐亚诺博士是艾滋病毒相关临床试验的专家。此外，还将开发一个核酸扩增/定量组件，实现样本到答案的完整操作，并进一步减少艾滋病毒病毒负荷检测的障碍。

DESCRIPTION (provided by applicant): Economic, logistical, and physical barriers limit HIV viral load quantification, the gold standard for HIV diagnostics. The cost of the test is high (approximately $100 per test) and the intricate equipment required limits testing to laboratories staffed by highly trained technicians. Also, the labile nature of the biomarker, viral RNA, require testing to be performed within 6 hours of blood draw unless the RNA can be stabilized, thus limiting the geographical reach of this test. Current point-of-care (POC) HIV tests rely on antibody titer, but this biomarker is limited in early, acute-stage infections, resulting in many false negatives. Additionally, strategies to preserve blood sample (e.g., dried blood spots) add additional steps to the workflow (e.g., spotting, drying, elution), resulting in added labor and increased potential for error. We propose a device that merges a novel blood collection method in commercial development by Tasso Inc., with a technology for sample preparation and stabilization developed at UW - Madison. Through a simple-to-use method integrating blood collection and sample preparation, the device will alleviate many of the barriers (time, cost, trained specialist) to the applications of HIV diagnostics. Further, a novel method for sample stabilization enables this method to be used and transported from any number of low-resource settings, expanding the reach of blood diagnostic methods. To achieve this project, Tasso is uniquely positioned through three key collaborations: 1) Professor David Beebe as an expert consultant for simple microfluidic devices for sample preparation with startup experience in 4 different companies, 2) Dr. Frank Graziano as an expert consultant for diagnostics for low-resource settings with a special affiliation with the Joint Clinical Research Centre (JCRC) in Uganda, and 3) the Morgridge Institutes for Research, enabling Tasso to have direct access to established prototyping facilities, along with access to experts in device design for manufacturability (including Thomas Mackie, founder of TomoTherapy). The proposal consists of three Aims. First, the Tasso device for blood acquisition will be modified for economically-limited deployment by making the non-blood contacting portion of the device reusable. Next, a one-step sample preparation cartridge, the SLIDE, will be optimized to extract viral RNA from blood samples collected with the Tasso device. Lastly, a wax-based sealing mechanism will be integrated into the SLIDE to enable on-chip reverse transcription, which stabilizes viral RNA into DNA, and sealing for transport. At the conclusion of Phase I, an integrated, optimized proof-of-concept platform will be capable of blood acquisition, viral RNA extraction, and RNA stabilization and storage. In Phase II, this prototype will be tested on patients with the assistance of consultant Dr. Frank Graziano, an expert in HIV- related clinical trials. Additionall, a nucleic acid amplification / quantitation component will be developed, enabling complete sample-to-answer operation and further mitigating barriers to HIV viral load testing access.