Department of Materials Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang, 37673, South Korea.
Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), Ulsan, 44429, South Korea.
Nanoscale. 2022 Jun 1;14(21):7828-7836. doi: 10.1039/d1nr07894a.
As DNA polymerases are even active at ambient temperature, there is inevitable non-specific amplification; to avoid the undesired amplification of analytes, a heat activation-based polymerase chain reaction (PCR), called hot-start PCR, is widely used to be highly precise and quantitative in detection. Unlike thermocycling amplification, isothermal amplification, compatible for point-of-care (PoC) tests, cannot be benefited by the heat-activation technique, making the method qualitative rather than quantitative. In this work, we newly developed a lead ion (Pb) activation technique, called lead-start isothermal amplification, allowing on-demand activation or deactivation of DNA polymerases at room temperature. We systematically correlated the DNA polymerase inhibition by the TQ30 aptamer with Pb-responsive strand cleavage by the GR5 DNAzyme, and relying on the type of interconnectors, Pb successfully served as an initiator or a terminator of isothermal DNA amplification. Our lead-start isothermal amplification was exceptionally Pb-specific, dramatically increasing the enzymatic activity of DNA polymerase (>25 times) only by Pb introduction. Despite one-by-one sample preparation, a number of reactions can begin and end at the same time, sharing the identical amplification conditions, and thereby allowing their quantitative analysis and comparison. Using a portable UV lamp and a smartphone camera, we also succeeded in quantifying the amounts of clinically important and human papillomavirus type 16 genes in human serum and SARS-CoV-2's nucleocapsid genes in human serum and saliva, and the limit of detection was as low as 0.1 nM, highly applicable for actual PoC tests in the field with no purification process.
由于 DNA 聚合酶在环境温度下也具有活性，因此不可避免地会发生非特异性扩增；为了避免分析物的非预期扩增，一种基于热激活的聚合酶链反应（PCR），称为热启动 PCR，被广泛用于实现高度精确和定量的检测。与热循环扩增不同，适用于即时检测（PoC）的等温扩增不能受益于热激活技术，因此该方法是定性的而不是定量的。在这项工作中，我们新开发了一种铅离子（Pb）激活技术，称为铅启动等温扩增，允许在室温下按需激活或失活 DNA 聚合酶。我们系统地将 TQ30 适体对 DNA 聚合酶的抑制作用与 GR5 DNA 酶对 Pb 响应链的切割作用相关联，并依赖于连接子的类型，Pb 成功地作为等温 DNA 扩增的启动子或终止子。我们的铅启动等温扩增具有出色的 Pb 特异性，仅通过引入 Pb 就可使 DNA 聚合酶的酶活性提高 25 倍以上。尽管逐个进行了样品制备，但多个反应可以同时开始和结束，共享相同的扩增条件，从而可以对其进行定量分析和比较。使用便携式 UV 灯和智能手机摄像头，我们还成功地定量了人血清中临床重要的和人乳头瘤病毒 16 基因以及人血清和唾液中 SARS-CoV-2 的核衣壳基因的含量，检测限低至 0.1 nM，非常适用于现场无纯化过程的实际 PoC 测试。
