Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 10²-10⁵ copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing.

中文翻译：

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一种基于纸的设备，用于实时定量检测多个DNA靶标进行环介导的等温扩增

纸质诊断设备具有多种优点，因为它具有简单性，便携性和成本效益，因此可作为即时诊断（POC）测试的多个诊断测试平台之一，具有许多优势。但是，尽管核酸测试（NAT）具有很高的灵敏度和特异性，但是基于纸质平台的NAT的开发却没有其他平台取得进展，因为核酸扩增反应的各种特定条件（例如pH，缓冲液成分和温度） ，抑制了基于纸张的设备的技术差异。在这里，我们提出了一种基于纸的设备，用于执行环介导的等温扩增（LAMP），同时实时检测多个DNA靶标。我们确定了最佳的化学成分，以使LAMP反应能够在没有冻干或其他技术的条件下达到干燥条件。我们还通过依次堆叠功能层设计了简单的纸设备结构，并利用了羟基萘酚蓝荧光的新发现的特性来分析纸设备中的实时LAMP信号。这个建议的平台允许在一个步骤中通过单步操作对三种不同的脑膜炎DNA样品进行分析。这种基于LAMP的多重诊断设备具有实时分析潜力，可定量检测10种² -10 ⁵份基因组DNA。此外，我们建议将DNA扩增设备转换为一种简单且负担得起的纸张系统方法，这对于实现基于纸张的NAT系统进行POC测试具有巨大的潜力。