移液管
核酸
核糖核酸
核酸法
检出限
DNA
色谱法
RNA提取
固相萃取
环介导等温扩增
化学
DNA提取
样品制备
生物
分子生物学
聚合酶链反应
生物化学
基因
物理化学
作者
Wenjing Lü,Jidong Wang,Qiong Wu,Jiashu Sun,Yiping Chen,Zifeng Lü,Chunsheng Zheng,Wenna Gao,Yi Liu,Xingyu Jiang
标识
DOI:10.1016/j.bios.2015.08.016
摘要
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.
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