V-OLA: point-of-care HIV viral load monitoring and drug resistance testing

摘要 我们将开发第一种检测病毒载量(VL)和艾滋病毒耐药性(DR)的方法，适用于 低资源设置(LRS)。这项新测试是基于多年开发和测试 HIV DR的寡核苷酸连接分析(OLA)，以及我们最近开发OLA-SIMPLE-A实验室试剂盒的工作 这减少了OLA的时间和复杂性，但仍然需要实验室设备，实验室单向 防止放大器污染的工作流程，以及多个步骤的实际操作。建议的V- OLA测试是OLA-SIMPLE的一个重大进步，它使用创新的技术来协调，以提供低成本， 简化了适合艾滋病毒患者管理的测试套件。我们将从以下方面开发快速样品制备 临床相关阈值的全血、半定量VL测量(目前WHO对 治疗失败是&gt；1000拷贝/毫升)，以及用于识别HIV DR突变的集成测试盒(目标是 检测VL&GT患者的10%突变；1000拷贝/毫升)。在目标1中，我们将发展化学和样品 HIV RNA分离的准备格式与快速VL检测兼容。在目标2中，我们将开发VL测试 采用半定量等温放大，包括简单、低成本的热阻和检测器。在《目标3》中， 我们将发展核心OLA化学，包括等温扩增DR靶区，连接单个 链式扩增子，以及用于扩增和OLA的干试剂；我们将对不同的HIV亚型进行检测。在……里面 目的4、我们将开发包括等温扩增和连接在内的集成DR检测装置 侧向流条带检测。在目标5中，我们将把VF和DR测试合并为一个协调系统，包括 完善硬件，扩大一次性套件的制造，并开发一种数字辅助系统，称为 水族馆-提供循序渐进的指导方案、样品跟踪、结果解释和计划 质量控制测试。我们将在储存的HIV血浆样本(HIV-1 A、C、D和AE亚型)上检测V-OLA。 这一级别的测试旨在为深入研究性能、可用性、 并付诸实施。

ABSTRACT We will develop the first test for detection of viral load (VL) and HIV drug resistance (DR) that is appropriate for low-resource settings (LRS). This new test is based on years of experience developing and testing the Oligonucleotide Ligation Assay (OLA) for HIV DR, as well as our recent work to develop OLA-Simple – a lab kit that reduces the time and complexity of OLA, but still requires laboratory equipment, laboratory one-way workflow to prevent contamination by amplicons, and hands-on operation of multiple steps. The proposed V- OLA test is a major advance over OLA-Simple that uses innovative technologies coordinated to give a low-cost, streamlined test suite appropriate for HIV patient management. We will develop rapid sample preparation from whole blood, semi-quantitative VL measurement at clinically-relevant thresholds (current WHO cut-off for treatment failure is >1000 copies/mL), and an integrated test cartridge to identify HIV DR mutations (goal to detect 10% mutant for patients with VL >1000 copies/mL). In Aim 1, we will develop chemistry and sample preparation format for HIV RNA isolation compatible with a rapid VL test. In Aim 2, we will develop the VL test using semi-quantitative isothermal amplification, including a simple low-cost heat block and detector. In Aim 3, we will develop core OLA chemistry including isothermal amplification of DR target regions, ligation on single- stranded amplicon, and dry reagents for amplification and OLA; we will test assays on different HIV subtypes. In Aim 4, we will develop the integrated DR detection device, including isothermal amplification and ligation with lateral flow strip detection. In Aim 5, we will combine the VF and DR tests into a coordinated system including refining hardware, scaling up fabrication of kit disposables, and develop a digitally-assisted system – called Aquarium – to provide step-by-step guided protocols, sample tracking, results interpretation, and scheduled quality control tests. We will test V-OLA on banked HIV plasma samples (HIV-1 subtypes of A, C, D, and AE). This level of testing is intended to provide preliminary evidence for in-depth studies on performance, usability, and implementation.