一步法核酸纯化和抗噪聚合酶链反应通过电动浓缩用于超低丰度核酸检测。
One-Step Nucleic Acid Purification and Noise-Resistant Polymerase Chain Reaction by Electrokinetic Concentration for Ultralow-Abundance Nucleic Acid Detection.
机构信息
Department of Electrical Engineering and Computer Science and Research Laboratory of Electronics, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA.
Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA.
出版信息
Angew Chem Int Ed Engl. 2020 Jun 26;59(27):10981-10988. doi: 10.1002/anie.201915788. Epub 2020 Apr 30.
Nucleic acid amplification tests (NAATs)integrated on a chip hold great promise for point-of-care diagnostics. Currently, nucleic acid (NA) purification remains time-consuming and labor-intensive, and it takes extensive efforts to optimize the amplification chemistry. Using selective electrokinetic concentration, we report one-step, liquid-phase NA purification that is simpler and faster than conventional solid-phase extraction. By further re-concentrating NAs and performing polymerase chain reaction (PCR) in a microfluidic chamber, our platform suppresses non-specific amplification caused by non-optimal PCR designs. We achieved the detection of 5 copies of M. tuberculosis genomic DNA (equaling 0.3 cell) in real biofluids using both optimized and non-optimal PCR designs, which is 10- and 1000-fold fewer than those of the standard bench-top method, respectively. By simplifying the workflow and shortening the development cycle of NAATs, our platform may find use in point-of-care diagnosis.
芯片上的核酸扩增测试（NAATs）在即时诊断方面具有巨大的应用前景。目前，核酸（NA）纯化仍然费时费力，并且需要大量的努力来优化扩增化学。我们通过选择性电泳浓缩，报告了一种一步式、液相 NA 纯化方法，比传统的固相萃取更简单、更快。通过进一步浓缩 NAs 并在微流控室中进行聚合酶链反应（PCR），我们的平台抑制了非最佳 PCR 设计引起的非特异性扩增。我们使用优化和非优化的 PCR 设计在真实生物流体中检测到 5 个拷贝的结核分枝杆菌基因组 DNA（相当于 0.3 个细胞），分别比标准台式方法少 10 倍和 1000 倍。通过简化工作流程和缩短 NAAT 的开发周期，我们的平台可能会在即时诊断中得到应用。
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