High-throughput sample-to-answer detection of DNA/RNA in crude samples within functionalized micro-pipette tips
High-throughput sample-to-answer detection of DNA/RNA in crude samples within functionalized micro-pipette tips
复制标题
在功能化微量移液器吸头内对粗样品中的 DNA/RNA 进行高通量样品到答案检测。
DOI：
10.1016/j.bios.2015.08.016
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发表时间：
2016-01-15
影响因子：
12.6
通讯作者：
Jiang, Xingyu
中科院分区：
文献类型：
--
作者：
Lu, Wenjing;Wang, Jidong;Jiang, Xingyu
我们开发了一种基于微量移液器吸头的核酸检测（MTNT），用于高通量的样品到答案检测来自包括细胞，细菌和固体植物的粗样品的DNA和RNA，而不需要样品预处理和复杂的操作。MTNT由微量移液器吸头和嵌入式固相核酸提取膜组成，完全集成了从粗样品中提取核酸、核酸的环介导等温扩增（LAMP）和检测的可视化读出的功能。MTNT的检出限为2个拷贝的含有埃博拉病毒靶核酸片段的质粒和8 CFU的携带埃博拉病毒衍生质粒的大肠杆菌。MTNT还可以检测CK-19 mRNA从少至2个癌细胞没有复杂的程序，如RNA提取和纯化。我们进一步证明MTNT在高通量格式使用八通道移液器和自制的迷你加热器，最大吞吐量为40个样品。与其他护理点（POC）核酸检测（NAT）相比，MTNT可以直接从液体（细胞/细菌/血液）或固体（植物）样本中以简单，灵敏，高通量和无污染的方式检测DNA和RNA，这表明在偏远地区进行低成本和POC NAT具有相当大的前景。(C)2015 Elsevier B. V.版权所有。
We develop a micro-pipette tip-based nucleic acid test (MTNT) for high-throughput sample-to-answer detection of both DNA and RNA from crude samples including cells, bacteria, and solid plants, without the need of sample pretreatment and complex operation. MTNT consists of micro-pipette tips and embedded solid phase nucleic acid extraction membranes, and fully integrates the functions of nucleic acid extraction from crude samples, loop-mediated isothermal amplification (LAMP) of nucleic acids, and visual readout of assays. The total assaying time for DNA or RNA from a variety of crude samples ranges from 90 to 160 min, The limit of detection (LOD) of MTNT is 2 copies of plasmids containing the target nucleic acid fragments of Ebola virus, and 8 CFU of Escherichia coli carrying Ebola virus-derived plasmids. MTNT can also detect CK-19 mRNA from as few as 2 cancer cells without complicated procedures such as RNA extraction and purification. We further demonstrate MTNT in a high-throughput format using an eight-channel pipette and a homemade mini-heater, with a maximum throughput of 40 samples. Compared with other point-of-care (POC) nucleic acid tests (NAT), MTNT could assay both DNA and RNA directly from liquid (cells/bacteria/blood) or solid (plant) samples in a straightforward, sensitive, high-throughput, and containment-free manner, suggesting a considerable promise for low-cost and POC NAT in remote areas. (C) 2015 Elsevier B.V. All rights reserved.