NAIL: Nucleic Acid detection using Isotachophoresis and Loop-mediated isothermal amplification
NAIL: Nucleic Acid detection using Isotachophoresis and Loop-mediated isothermal amplification
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DOI：
10.1039/c4lc01479k
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发表时间：
2015-01-01
期刊：
影响因子：
6.1
通讯作者：
Posner, Jonathan D.
中科院分区：
文献类型：
--
作者：
Borysiak, Mark D.;Kimura, Kevin W.;Posner, Jonathan D.
核酸扩增检测是许多传染病诊断的金标准，因为它具有高灵敏度和特异性、快速操作和低检测限。尽管核酸扩增测试具有优势，但由于需要复杂的仪器和费力的样品制备，它们目前提供有限的护理点（POC）效用。我们报道了核酸等速电泳LAMP（NAIL）诊断装置的研制。NAIL使用等速电泳（ITP）和环介导等温扩增（LAMP）在集成芯片内不到一小时的时间内从复杂基质中提取和扩增核酸。ITP是一种电动分离技术，其使用电场和两种缓冲液在单个步骤中提取和纯化核酸。LAMP在恒定温度下扩增核酸，并产生大量易于检测的DNA。移动的电话对放大结果进行成像，以消除对激光荧光检测的需要。该设备需要最少的用户干预，因为毛细管阀和加热的空气室作为被动阀和泵自动流体驱动。在本文中，我们描述了NAIL装置的设计和操作，并演示了致病性E。大肠杆菌O 157：H7细胞。我们使用临床和实验室标准化协会（CLSI）的检测限（LoD）定义，该定义考虑了阳性和阴性样本的方差，以确定诊断LoD。根据CLSI定义，NAIL器械对大肠杆菌的检出限（LoD）为1000 CFU mL（-1）。大肠杆菌细胞人工接种到全脂牛奶，这是两个数量级的改进，以标准管LAMP反应稀释牛奶样品和基于实验室的方法。NAIL设备有可能显著降低POC应用中传统核酸诊断的复杂性和成本。
Nucleic acid amplification tests are the gold standard for many infectious disease diagnoses due to high sensitivity and specificity, rapid operation, and low limits of detection. Despite the advantages of nucleic acid amplification tests, they currently offer limited point-of-care (POC) utility due to the need for complex instruments and laborious sample preparation. We report the development of the Nucleic Acid Isotachophoresis LAMP (NAIL) diagnostic device. NAIL uses isotachophoresis (ITP) and loop-mediated isothermal amplification (LAMP) to extract and amplify nucleic acids from complex matrices in less than one hour inside of an integrated chip. ITP is an electrokinetic separation technique that uses an electric field and two buffers to extract and purify nucleic acids in a single step. LAMP amplifies nucleic acids at constant temperature and produces large amounts of DNA that can be easily detected. A mobile phone images the amplification results to eliminate the need for laser fluorescent detection. The device requires minimal user intervention because capillary valves and heated air chambers act as passive valves and pumps for automated fluid actuation. In this paper, we describe NAIL device design and operation, and demonstrate the extraction and detection of pathogenic E. coli O157:H7 cells from whole milk samples. We use the Clinical and Laboratory Standards Institute (CLSI) limit of detection (LoD) definitions that take into account the variance from both positive and negative samples to determine the diagnostic LoD. According to the CLSI definition, the NAIL device has a limit of detection (LoD) of 1000 CFU mL(-1) for E. coli cells artificially inoculated into whole milk, which is two orders of magnitude improvement to standard tube-LAMP reactions with diluted milk samples and comparable to lab-based methods. The NAIL device potentially offers significant reductions in the complexity and cost of traditional nucleic acid diagnostics for POC applications.