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利用环介导等温扩增增强的粒子扩散测量法实时可视化 HIV-1 RNA 检测。

Real-Time Visualization of HIV-1 RNA Detection Using Loop-Mediated Isothermal Amplification-Enabled Particle Diffusometry.

机构信息

School of Mechanical Engineering, Purdue University, West Lafayette, Indiana 47907, United States.

Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana 47907, United States.

出版信息

ACS Sens. 2024 Oct 25;9(10):5541-5549. doi: 10.1021/acssensors.4c01870. Epub 2024 Oct 8.

Abstract

Isothermal nucleic acid amplification tests, NAATs, such as reverse transcription-loop-mediated isothermal amplification (RT-LAMP), offer promising capabilities to perform real-time semiquantitative detection of viral pathogens. These tests provide rapid results, utilize simple instrumentation for single-temperature reactions, support efficient user workflows, and are suitable for field use. Herein, we present a novel and robust method for real-time monitoring of HIV-1 RNA RT-LAMP utilizing a novel implementation of particle diffusometry (PD), a diffusivity quantification technique using fluorescent particles, to quantify viral concentration in nuclease-free water. We monitor changes in particle diffusion dynamics of 400 nm fluorescently labeled particles throughout the RT-LAMP of HIV-1 RNA in nuclease-free water, enabling measurement within 20 min and detection of concentrations as low as 25 virus particles per μL. Moreover, in a single-blind study, we demonstrate semiquantitative detection by accurately determining the initial concentration of an unknown HIV-1 RNA within a 10% absolute error margin. These results highlight the potential of real-time PD readout for quantifying HIV-1 RNA via RT-LAMP, offering promise for viral load monitoring of HIV and other chronic infections.

摘要

等温核酸扩增检测(NAATs),如逆转录环介导等温扩增(RT-LAMP),提供了有前途的实时半定量检测病毒病原体的能力。这些测试提供快速的结果,使用简单的仪器进行单温度反应,支持高效的用户工作流程,并且适合现场使用。在这里,我们提出了一种新颖而强大的方法,用于实时监测 HIV-1 RNA RT-LAMP,利用粒子扩散度测定法(PD)的新实现,这是一种使用荧光粒子进行扩散度定量的技术,用于定量无核酸酶水中的病毒浓度。我们监测无核酸酶水中 HIV-1 RNA 的 RT-LAMP 过程中 400nm 荧光标记粒子的扩散动力学变化,能够在 20 分钟内进行测量,并检测低至 25 个病毒粒子/μL 的浓度。此外,在一项单盲研究中,我们通过准确确定未知 HIV-1 RNA 的初始浓度,在 10%的绝对误差范围内,证明了半定量检测。这些结果突出了实时 PD 读数在通过 RT-LAMP 定量 HIV-1 RNA 方面的潜力,为 HIV 和其他慢性感染的病毒载量监测提供了希望。

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