A self-contained microfluidic in-gel loop-mediated isothermal amplification for multiplexed pathogen detection
A self-contained microfluidic in-gel loop-mediated isothermal amplification for multiplexed pathogen detection
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DOI：
10.1016/j.snb.2016.07.164
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发表时间：
2017-02-01
影响因子：
8.4
通讯作者：
Liu, Bi-Feng
中科院分区：
文献类型：
--
作者：
Chen, Chen;Liu, Pian;Liu, Bi-Feng
在这项工作中，我们提出了一种微流体凝胶环介导的等温扩增（gLAMP）包含预装的试剂作为一个独立的微型设备，用于同时检测多个致病性细菌DNA在近点的护理设置。微芯片由多个通道组成，反应室容纳低熔点琼脂糖和除DNA样品外的所有LAMP试剂，DNA样品可在4 ℃下长期保存。目前的微芯片可以同时测试四种不同的细菌DNA靶标。将DNA样品引入微芯片后，在65 ℃的热板上进行gLAMP反应。进一步优化了gLAMP反应条件，包括荧光指示剂、Bst DNA聚合酶、引物和Mg 2+离子的浓度。在优化的条件下，对血清样品中的大肠杆菌、豪泽变形杆菌、副溶血性弧菌和沙门氏菌等食源性致病菌进行了检测。肠杆菌的选择性和灵敏度高（低至3个拷贝/μ L）。gLAMP微芯片为基于LAMP的病原体检测提供了一个简单且易于操作的平台，该平台在未来的护理点环境中具有很高的应用潜力。(C)© 2016 Elsevier B. V.版权所有。
In this work, we propose a microfluidic in-gel loop-mediated isothermal amplification (gLAMP) containing preloaded reagents as a self-contained microdevice for simultaneous detection of multiple pathogenic bacterial DNA in near point of care settings. The microchip consists of multiple channels with reaction chambers accommodating low-melting-point agarose and all LAMP reagents except the DNA samples, which can be kept at 4 degrees C for long-term storage. The current microchip enables simultaneous tests of four different bacterial DNA targets. After DNA samples were introduced into the microchip, gLAMP reactions were carried out on a hot plate at 65 degrees C. The gLAMP reaction conditions including the concentrations of the fluorescence indicator, Bst DNA polymerase, primers, and Mg2+ ions were further optimized. Under the optimized conditions, detection of foodborne bacteria in serum samples, including Escherichia coli, Proteus hauseri, Vibrio parahaemolyticus, and Salmonella subsp. Enterica were demonstrated with high selectivity and sensitivity (as low as 3 copies/mu L). The gLAMP microchip provides a simple and easy-to-operate platform for LAMP-based pathogen detection, which holds high potential for future applications in point-of-care settings. (C) 2016 Elsevier B.V. All rights reserved.