Department of Mechanical Engineering, Toyohashi University of Technology, Aichi 441-8580, Japan.
Institute for Research on Next-generation Semiconductor and Sensing Science (IRES2), Toyohashi University of Technology, Aichi 441-8580, Japan.
Analyst. 2024 Jun 10;149(12):3335-3345. doi: 10.1039/d4an00215f.
In this study, a microfluidic-based system utilizing colorimetric loop-mediated isothermal amplification (LAMP) is introduced for the quantitative analysis of nucleic acid targets. This system offers a user-friendly and cost-effective platform for the multiplexed genetic diagnosis of various infectious diseases across multiple samples. It includes time-lapse imaging equipment for capturing images of the microfluidic device during the LAMP assay and a hue-based quantitative analysis software to analyze the LAMP reaction, streamlining diagnostic procedures. An electric pipette was used to simplify the loading of samples and LAMP reagents into the device, allowing easy operation even by untrained individuals. The hue-based analysis software employs efficient image processing and post-processing techniques to calculate DNA amplification curves based on color changes in multiple reaction chambers. This software automates several tasks, such as identifying reaction chamber areas from time-lapse images, quantifying color information within each chamber, correcting baselines of DNA amplification curves, fitting experimental data to theoretical curves, and determining the threshold time for each curve. To validate the developed system, conventional off-chip LAMP assays were conducted with a 25 μL reaction mixture in 0.2 mL polymerase chain reaction (PCR) tubes using a real-time turbidimeter. The results indicated that the threshold time obtained using the colorimetric LAMP assay in the developed system is comparable to that obtained with real-time turbidity measurements in PCR tubes, demonstrating the system's capability for quantitative analysis of target nucleic acids, including those from human herpesviruses.
在这项研究中，引入了一种基于微流控的系统，利用比色环介导的等温扩增（LAMP）进行核酸靶标定量分析。该系统为多种传染病的多样本基因诊断提供了一种用户友好且经济高效的平台。它包括用于捕获 LAMP 测定期间微流体设备图像的时程成像设备，以及基于色调的定量分析软件，用于分析 LAMP 反应，简化诊断程序。电动移液器用于简化样品和 LAMP 试剂向设备中的加载，即使是非专业人员也可以轻松操作。基于色调的分析软件采用高效的图像处理和后处理技术，根据多个反应室中的颜色变化计算 DNA 扩增曲线。该软件自动执行多项任务，例如从时程图像中识别反应室区域，量化每个室中的颜色信息，校正 DNA 扩增曲线的基线，拟合实验数据与理论曲线，并确定每条曲线的阈值时间。为了验证开发的系统，使用实时浊度计在 0.2 mL 聚合酶链反应（PCR）管中用 25 μL 反应混合物进行了常规的离芯片 LAMP 测定。结果表明，在开发系统中进行比色 LAMP 测定得到的阈值时间与在 PCR 管中进行实时浊度测量得到的阈值时间相当，证明了该系统对目标核酸进行定量分析的能力，包括来自人类疱疹病毒的核酸。
