Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids
Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids
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DOI：
10.1039/c1an15289k
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发表时间：
2011-01-01
期刊：
影响因子：
4.2
通讯作者：
Marchal, Damien
中科院分区：
文献类型：
--
作者：
Kivlehan, Francine;Mavre, Francois;Marchal, Damien
我们描述了一种电化学方法来实时监测核酸的等温解旋酶依赖性扩增。检测原理简单，非常适合便携式、易于使用和廉价的核酸检测技术的发展。它包括在等温DNA扩增过程中监测报告DNA嵌入氧化还原探针的电化学电流响应的降低。该方法提供了在单一恒定温度下在不到一小时内定量分析靶核酸的可能性，并在等温扩增结束时进行DNA熔解曲线分析，以区分特异性和非特异性扩增。为了说明这种方法用于开发具有高通量能力的简单，稳健和低成本仪器的潜力，该方法用能够在由48个电化学微孔组成的一次性微孔板中同时监测多达48个实时等温HDA反应的电化学系统进行了验证。用这种方法获得的结果与一个完善的，但更复杂和昂贵的荧光为基础的方法获得的结果是相当的。这使得一个有前途的替代检测方法，不仅用于实时等温解旋酶依赖性扩增的核酸，但也为其他等温DNA扩增策略。
We described an electrochemical method to monitor in real-time the isothermal helicase-dependent amplification of nucleic acids. The principle of detection is simple and well-adapted to the development of portable, easy-to-use and inexpensive nucleic acids detection technologies. It consists of monitoring a decrease in the electrochemical current response of a reporter DNA intercalating redox probe during the isothermal DNA amplification. The method offers the possibility to quantitatively analyze target nucleic acids in less than one hour at a single constant temperature, and to perform at the end of the isothermal amplification a DNA melt curve analysis for differentiating between specific and nonspecific amplifications. To illustrate the potentialities of this approach for the development of a simple, robust and low-cost instrument with high throughput capability, the method was validated with an electrochemical system capable of monitoring up to 48 real-time isothermal HDA reactions simultaneously in a disposable microplate consisting of 48-electrochemical microwells. Results obtained with this approach are comparable to that obtained with a well-established but more sophisticated and expensive fluorescence-based method. This makes for a promising alternative detection method not only for real-time isothermal helicase-dependent amplification of nucleic acid, but also for other isothermal DNA amplification strategies.