Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids |
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Authors: | Kivlehan Francine Mavré François Talini Luc Limoges Benoît Marchal Damien |
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Affiliation: | Laboratoire d'Electrochimie Moléculaire, UMR 7591 CNRS, Univ Paris Diderot, Sorbonne Paris Cité, 15 rue Jean-Antoine de Ba?f, F-75205 Paris Cedex 13, France. |
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Abstract: | 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 non-specific 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. |
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