PDF(Pubmed)
Abstract：
Nucleic acid amplification testing has great potential for point-of-need diagnostic testing with high detection sensitivity and specificity. Current sample preparation is limited by a tedious workflow requiring multiple steps, reagents and instrumentation, hampering nucleic acid testing at point of need. In this study, we present the use of mixed cellulose ester (MCE) paper for DNA binding by ionic interaction under molecular crowding conditions and fluid transport by wicking. The poly(ethylene) glycol-based (PEG) reagent simultaneously provides the high pH for alkaline lysis and crowding effects for ionic binding of the DNA under high salt conditions. In this study, we introduce Paper-based Abridged Solid-Phase Extraction with Alkaline Poly(ethylene) Glycol Lysis (PASAP). The anionic mixed cellulose ester (MCE) paper is used as solid phase and allows for fluid transport by wicking, eliminating the need for pipetting skills and the use of a magnet to retain beads. Following the release of DNA from the cells due to the lytic activity of the PASAP solution, the DNA binds to the anionic surface of the MCE paper, concentrating at the bottom while the sample matrix is transported towards the top by wicking. The paper was washed by dipping it in 40% isopropanol for 10 s. After air-drying for 30 s, the bottom section of the paper (3 mm × 4 mm) was snapped off using the cap of a PCR tube and immersed in the colourimetric loop-mediated isothermal amplification (cLAMP) solution for direct amplification and colourimetric detection. The total sample processing was completed in 15 min and ready for amplification. cLAMP enabled the detection of 102 CFU/mL of Escherichia coli (E. coli) from culture media and the detection of E. coli in milk < 103 CFU/mL (10 CFU) after incubation at 68 °C for 60 min, demonstrating applicability of the method to complex biological samples.
摘要：
核酸扩增测试具有高检测灵敏度和特异性的需求点诊断测试的巨大潜力。当前的样品制备受到需要多个步骤的繁琐工作流程的限制,试剂和仪器，在需要的时候阻碍核酸测试。在这项研究中,我们介绍了混合纤维素酯（MCE）纸在分子拥挤条件下通过离子相互作用与DNA结合以及通过芯吸进行流体传输的用途。基于聚(乙二醇)二醇(PEG)的试剂在高盐条件下同时提供用于碱解的高pH和用于DNA的离子结合的拥挤效应。在这项研究中,我们介绍了基于碱性聚乙二醇裂解（PASAP）的纸基简化固相萃取。阴离子混合纤维素酯（MCE）纸用作固相，并允许通过芯吸，无需移液技巧和使用磁铁来保留珠子。由于PASAP溶液的裂解活性，DNA从细胞中释放后，DNA结合到MCE纸的阴离子表面，在底部浓缩,而样品基质通过芯吸朝向顶部输送。通过将纸浸入40%异丙醇中10秒来洗涤。在空气干燥30秒后,使用PCR管帽将纸的底部（3mm×4mm）折断，并浸入比色环介导等温扩增（cLAMP）溶液中，以进行直接扩增和比色检测。总样品处理在15分钟内完成并准备用于扩增。cLAMP能够检测102CFU/mL的大肠杆菌(E.大肠杆菌）来自培养基，并在68°C孵育60分钟后检测牛奶中的大肠杆菌<103CFU/mL（10CFU），证明该方法适用于复杂的生物样品。
