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