Rapid, quantitative isothermal molecular assay for POC HIV-1 viral load monitoring using amplification nucleation site analysis

项目摘要 超过2000万艾滋病毒携带者正在接受抗逆转录病毒治疗，需要携带艾滋病毒。 检测以确定病毒学失败的病例并提供可操作的信息来指导替代临床 治疗。目前检测HIV病毒载量的方法依赖于定量聚合酶链式反应(QPCR)或数字聚合酶链式反应 (DPCR)，这通常限于资源丰富的中央实验室，需要 分散艾滋病毒病毒载量监测，以实现快速、基于诊所或家庭自我检测的病毒载量测量。 我们已经确定了一种实现数字等温放大的新方法，该方法利用 重组酶聚合酶扩增(RPA)等温扩增的特征粘性反应缓冲液 化学和商业上可用的多孔膜。我们建议应用扩增成核位点 用于HIV-1病毒载量监测的分析(ANSA)，以准确量化与临床相关的HIV-1 RNA 亚型和病毒载量。我们提出了两个探索性目标，以证明ANSA可以实现所需的 HIV-1亚型病毒载量动态范围和定量精确度。

Project Summary Over 20 million people living with HIV (PLHIV) are receiving antiretroviral therapy and require HIV viral load testing to identify cases of virological failure and provide actionable information to guide alternative clinical treatment. Current methods for HIV viral load measurement rely on quantitative PCR (qPCR) or digital PCR (dPCR), which are commonly restricted to highly resourced central laboratories and there is a need to decentralize HIV viral load monitoring to enable rapid, clinic-based or home self-testing viral load measurements. We have identified a novel method for implementing digital isothermal amplification that leverages the characteristic viscous reaction buffer of recombinase polymerase amplification (RPA) isothermal amplification chemistry and commercially available porous membranes. We propose to apply amplification nucleation site analysis (ANSA) for HIV-1 viral load monitoring to accurately quantify HIV-1 RNA over clinically relevant HIV-1 subtypes and viral loads. We propose two exploratory aims to demonstrate that ANSA can achieve the required viral load dynamic range and quantitative precision across HIV-1 subtypes.