We have developed a novel paper-based molecular pathogen diagnostic method by combining the direct loop-mediated isothermal amplification (Direct LAMP) in a paper reactor and an immunochromatographic strip (ICS) detection without any DNA preparation step. The mineral paper is durable, oil- and tear-resistant, and waterproof, so it is a proper material for point-of-care (POC) testing owing to facile chip fabrication, cost-effectiveness, and disposability. The mineral paper was utilized as a substrate to construct the reactor for gene amplification based on the principle of origami without using an expensive and complicated process. In this novel paper reactor, we performed the Direct LAMP reaction, which is a direct isothermal DNA amplification without any sample preparation step such as cell lysis, DNA extraction and purification for amplifying specific target gene. After the Direct LAMP reaction in a paper reactor, a capillary tube was employed to take an appropriate amount (~ 2 μL) of amplicons in order to transfer the LAMP product to an ICS. The use of the ICS allowed us to perform the simple and rapid colorimetric detection of the resultant amplified target genes by naked eyes, thereby confirming the presence of the pathogen in a sample in a user-friendly way. In this platform, we could simultaneously identify Staphylococcus aureus and Escherichia coli O157:H7 contaminated in human blood or milk. The whole molecular diagnostic process (the Direct LAMP in a mineral paper plus the ICS detection) could be conducted in a paper substrate, so the proposed method would be ideal for pathogen detection in resourcelimited environments.

中文翻译：

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无需样品制备步骤即可从人全血和牛奶中扩增和检测病原细菌的基于纸的分子诊断

我们已经开发了一种新颖的基于纸张的分子病原体诊断方法，该方法将纸张反应器中的直接环介导的等温扩增（Direct LAMP）与无需任何DNA制备步骤的免疫色谱带（ICS）检测相结合。矿物纸坚固耐用，耐油和抗撕裂且防水，因此由于芯片制造容易，成本效益高和可处置性强，因此它是即时检验（POC）测试的合适材料。根据折纸原理，使用矿物纸作为基质来构建用于基因扩增的反应器，而无需使用昂贵且复杂的过程。在此新型纸质反应器中，我们进行了直接LAMP反应，这是直接等温DNA扩增，无需任何样品制备步骤，例如细胞裂解，DNA提取和纯化，用于扩增特定的靶基因。在纸反应器中进行直接LAMP反应后，使用毛细管取适量（〜2μL）的扩增子，以将LAMP产物转移至ICS。ICS的使用使我们能够通过肉眼对所得扩增的靶基因进行简单，快速的比色检测，从而以用户友好的方式确认样品中病原体的存在。在这个平台上，我们可以同时识别 ICS的使用使我们能够通过肉眼对所得扩增的靶基因进行简单，快速的比色检测，从而以用户友好的方式确认样品中病原体的存在。在这个平台上，我们可以同时识别 ICS的使用使我们能够通过肉眼对所得扩增的靶基因进行简单，快速的比色检测，从而以用户友好的方式确认样品中病原体的存在。在这个平台上，我们可以同时识别人血或牛奶中污染的金黄色葡萄球菌和大肠杆菌O157：H7。整个分子诊断过程（矿物纸中的直接LAMP加ICS检测）可以在纸质基材上进行，因此，该方法对于资源有限的环境中的病原体检测是理想的。