Novel Quantitative Cost-effective Assay for the Diagnosis and Monitoring of HIV viral loads

项目总结 自艾滋病流行开始以来，根据联合国艾滋病毒/艾滋病联合规划署(艾滋病规划署)， 共有7570万人感染艾滋病毒，其中3270万人死于艾滋病-- 相关疾病。2016年，艾滋病规划署确定了到2020年要实现的艾滋病毒检测和治疗的几个目标 到2030年结束艾滋病的流行。这些里程碑包括将年感染率降至500,000 每年新增感染人数，每年与艾滋病有关的死亡人数减少到每年500万人，以及90%- 90-90的目标，要求90%的艾滋病毒感染者了解他们的状态，其中90%正在接受 治疗，其中90%的病毒载量无法检测到。我们现在已经达到了目标年，并且基于 从2019年的数据来看，我们预计不会达到这些里程碑中的任何一个。尽管这些目标是在一个 少数国家，大多数低收入和中等收入国家继续落后于全球目标，部分原因是 资金和收入差距的缩小。为了实现这些目标，需要进行广泛的艾滋病毒检测，而不是 只是为了诊断新病例，但为了监测艾滋病毒携带者的病毒载量。因此，有一个 迫切需要一种可以在低资源环境下使用的特定病毒检测平台。在这里，我们建议 开发一种针对HIV-1和HIV-2的特异、敏感和快速检测的新技术，使用通用的 识别护理点测试平台。这一独特的平台集成了特定的电化学检测 使用四向连接(4WJ)探针对病毒基因组序列进行修改的等温扩增 基于核酸序列扩增(NASBA)的病毒RNA片段。 完整的分析(RNA扩增和检测)预计在不到一小时内执行， 可以与定期去医生办公室看病相媲美。在拟议的研究中，我们将：1)优化 基于NASBA的修正TAG等温扩增；3)通过选择合适的参数设计电化学生物传感器 4WJ探针要询问的适当的扩增片段；4)优化和表征 用于病毒直接检测扩增片段的生物传感器；5)单、多重生物传感器的可变参数研究 分析；6)将开发的生物传感器应用于商业参考材料的病毒检测；以及 7)使用参考方法(RT-qPCR)验证我们的方法。 这项拟议的技术有望成为一种在资源有限的情况下进行的用户友好的医疗点测试 及时诊断艾滋病毒感染并进行监测，帮助在2030年前遏制艾滋病感染的扩散。

PROJECT SUMMARY Since the start of the AIDS epidemic, according to the United Nations Joint Programme on HIV/AIDS (UNAIDS), a total of 75.7 million people have been infected with HIV, and of which 32.7 million have died due to AIDS- related illnesses. In 2016, UNAIDS defined several targets for HIV testing and treatment to be achieved by 2020 to end the AIDS epidemic by 2030. Such milestones include the reduction of the yearly infection rate to 500,000 new infections per year, the reduction of yearly AIDS-related deaths to 5000,000 deaths per year, and the 90- 90-90 goal, requiring that 90% of HIV-infected individuals be aware of their status, of which 90% are receiving treatment, and of which 90% have undetectable viral loads. We have now reached the goal year, and, based on data from 2019, we are not projected to meet any of these milestones. Although the targets were reached in a handful of countries, most low- and middle-income countries continue to fall behind the global goal in part due to decreases in funding and income disparities. To achieve these goals, extensive HIV testing is required, not only to diagnose new cases but in order to monitor the viral loads of those living with HIV. Therefore, there is an urgent need for a specific virus detection platform that can be used in low-resources settings. Here, we propose to develop a new technology for the specific, sensitive, and rapid detection of HIV-1 and HIV-2, using a universal recognition point-of-care testing platform. This unique platform integrates electrochemical detection of specific viral genome sequences using four-way junction (4WJ) probes following a modify isothermal amplification of the viral RNA fragments using Nucleic Acid Sequence Based Amplification (NASBA). The complete analysis (RNA amplification and detection) is expected to be executed in less than one hour, which is comparable with a regular visit to the doctor’s office. In the proposed research we will: 1) Optimized the modified-tag isothermal amplification using NASBA; 3) Design an electrochemical biosensor by selecting appropriate fragments of the amplicons to be interrogated by the 4WJ probes; 4) Optimize and characterize the biosensor for virus direct detection of amplicons; 5) Study variable parameters for single- and multiplex biosensor analysis; 6) Apply the developed biosensors for virus detection of commercially available reference material; and 7) Validate our methodology using a reference method (RT-qPCR). The proposed technology promises to be a user-friendly point-of-care testing at limited-resource settings for timely diagnosis of HIV infection and monitoring to help stopping the proliferation of AIDS infection by 2030.