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

艾滋病毒是导致艾滋病的病毒，它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携带者，并通过抗逆转录病毒治疗将他们的病毒控制到无法检测的水平，艾滋病毒就可以得到有效的管理。通过及早发现耐药性或治疗依从性差引起的病毒反弹，增加获得常规病毒载量检测的机会，有助于降低治疗失败率。为此，艾滋病毒自我检测，即希望了解艾滋病毒状况的个人收集样本、进行检测并私下解释结果的过程，已成为一种赋权和创新的方法。现有的HIV自我检测方法几乎完全依赖于基于横向流动的检测方法来检测宿主对HIV感染的抗体反应。在早期感染窗口的6-12周期间，他们可能会错过很大一部分无症状个体；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量化的唯一方法。然而，由于样品的处理和分析的复杂性，它的使用受到外行的限制。拟议的研究有可能显著提高抗逆转录病毒治疗下艾滋病毒感染者的治疗结果。此外，通过教育和推广活动，拟议的研究处于有利地位，以增强综合学习体验，并在多个层面吸引多学科的学生。这项建议的主要研究目标是探索一种在带有USB分析仪的一次性微流控芯片上的超紧凑定量核酸测试(NAT)，以检测HIV-1病毒反弹，这种测试足够简单，供外行测试自己以监测治疗依从性。其目标是开发和验证一种基于全血的测试，可以半定量地评估细胞浓度低至1000拷贝/毫升的HIV-1RNA的存在。该测试可以进行类似于家庭血糖测试的单步手指刺血加载。为此，将开展关于微流控样品制备、扩增分析、USB分析仪集成和分析验证的四项研究任务。任务1.自动微流控样品制备。将开发一种流线型微流控芯片，用于自动分离血浆和从全血中提取RNA。任务2。将优化HIV-1实时逆转录环介导的等温扩增分析，并探索检测的最低限度。任务3.分析器集成和小规模原型制作。将开发和集成分析仪硬件和软件，以实现简单和可靠的操作。任务4.对照样品的实验室验证。原型设备将在实验室环境中使用添加到全血中的HIV-1血浆样本进行验证。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.