Quantitative microfluidic NAT-on-USB: towards routine HIV viral load testing

导致艾滋病的艾滋病毒仍然是一个重大的公共卫生问题。幸运的是，如果能够识别艾滋病毒感染者，并通过抗逆转录病毒疗法将其病毒控制在无法检测的水平，就可以有效地控制艾滋病毒。增加常规病毒载量检测的可及性可以通过早期检测由耐药性或治疗依从性差引起的病毒反弹来帮助降低治疗失败率。为此，艾滋病毒自我检测，即想了解艾滋病毒状况的个人私下收集样本、进行检测并解释结果的过程，已成为一种增强权能的创新办法。现有的HIV自我检测方法几乎完全依赖于基于侧流的检测来检测宿主对HIV感染的抗体反应。他们可能会在6-12周的早期感染窗口期错过相当一部分无症状个体;他们也缺乏检测病毒反弹的能力。核酸检测是目前病毒载量定量的唯一方法。然而，由于样品处理和测定的复杂性，其由外行人使用是有限的。拟议的研究有可能显着提高抗逆转录病毒治疗下的艾滋病毒患者的治疗效果。此外，通过教育和外联活动，拟议的研究非常适合增强综合学习体验并吸引多个层面的多学科学生。该提案的主要研究目标是探索在一次性微流控芯片上进行超紧凑的定量核酸检测（NAT），配备USB分析仪来检测HIV-1病毒反弹，这是足够简单的外行测试自己监测治疗依从性。目标是开发和验证一种基于全血的检测方法，该方法可以在低至1000拷贝/ml的细胞浓度下半定量评估HIV-1 RNA的存在。该测试可以类似于家庭血糖测试进行，只需一步手指采血。为此，将进行关于微流控样品制备、扩增测定、USB分析仪集成和分析验证的四项研究任务。任务1.自动化微流体样品制备。将开发用于自动血浆分离和全血RNA提取的流线型微流控芯片。任务2.将优化HIV-1实时逆转录环介导等温扩增试验，并探索可能的最低检测限。任务3.分析仪集成和小规模原型设计。分析仪的硬件和软件将被开发和集成，以实现简单和可靠的操作。任务4.用对照样品进行实验室验证。该原型设备将在实验室环境中使用HIV-1血浆样本掺入全血进行验证。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

HIV, the virus that causes AIDS, continues to be a significant public health issue. Fortunately, HIV can be effectively managed if people of HIV can be identified and their virus is controlled to undetectable levels by antiretroviral therapy. Increasing access to routine viral load testing can help reduce the treatment failure rate by early detection of viral rebound caused by either drug resistance or poor therapy adherence. To this end, HIV self-testing, a process in which individual who wants to know HIV status collects a specimen, performs a test and interprets the result in private, has become an empowering and innovative approach. Existing HIV self-testing methods rely almost exclusively on lateral flow based test to detect host antibody response to HIV infection. They could miss a significant portion of asymptomatic individuals during the 6-12 weeks of early infection window; they also lack the ability for detection of viral rebound. Nucleic acid testing is currently the only method for viral load quantification. Nevertheless, its use by laypersons is limited due to the sample handling and assay complexity. The proposed research has the potential to significantly enhance the treatment outcomes for individuals of HIV under antiretroviral therapy. In addition, with educational and outreach activities, the proposed research is well positioned to enhance the integrative learning experience and to engage multidisciplinary students at many levels.The primary research objective of this proposal is to explore an ultra-compact quantitative nucleic acid testing (NAT) on a disposable microfluidic chip with a USB analyzer to detect HIV-1 viral rebound that is simple enough for laypersons to test themselves to monitor treatment adherence. The goal is to develop and validate a whole blood-based test that can semi-quantitatively assess the presence of HIV-1 RNA at cell concentrations as low as 1000 copies/ml. The test can be performed similarly to a home blood glucose test with a single step of finger-prick blood loading. To this end, four research tasks regarding the microfluidic sample preparation, amplification assay, USB analyzer integration, and analytical validation will be pursued. Task 1. Automated microfluidic sample preparation. A streamlined microfluidic chip for automated plasma separation and RNA extraction from whole blood will be developed. Task 2. An HIV-1 Real-time reverse transcription loop-mediated isothermal amplification assay will be optimized and the lowest possible limit of detection will be explored. Task 3. Analyzer integration and small-scale prototyping. The analyzer hardware and software will be developed and integrated for easy and robust operation. Task 4. Laboratory validation with control samples. The prototyped device will be validated in lab settings using HIV-1 plasma samples spiked into whole blood.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.