title: Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids.
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.
authors.affiliation: Laboratoire d'Electrochimie Moléculaire, UMR 7591 CNRS, Univ Paris Diderot, Sorbonne Paris Cité, 15 rue Jean-Antoine de Baïf, F-75205 Paris Cedex 13, France.
authors.forename: Francine
authors.initials: F
authors.lastname: Kivlehan
authors.forename: François
authors.initials: F
authors.lastname: Mavré
authors.forename: Luc
authors.initials: L
authors.lastname: Talini
authors.forename: Benoît
authors.initials: B
authors.lastname: Limoges
authors.forename: Damien
authors.initials: D
authors.lastname: Marchal
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism; D055664:Electrochemical Techniques / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D007364:Intercalating Agents / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques; D010084:Oxidation-Reduction; D044366:Transition Temperature
chemical_list: D015342:DNA Probes; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D004247:DNA; D004265:DNA Helicases