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Please use this identifier to cite or link to this item: http://hdl.handle.net/10651/32536

Title: Attomolar quantification of Mycobacterium tuberculosis by asymmetric helicase dependent isothermal DNA-amplification and electrochemical detection
Author(s): Barreda García, Susana
González Álvarez, María José
Santos Álvarez, Noemí de los
Palacios Gutiérrez, Juan José
Miranda Ordieres, Arturo José
Lobo Castañón, María Jesús
Issue date: 2015
Publisher: Elsevier
Publisher version: http://dx.doi.org/10.1016/j.bios.2014.12.029
Citation: Biosensors and Bioelectronics, 68, p. 122-128 (2014); doi:10.1016/j.bios.2014.12.029
Format extent: p. 122-128
Abstract: A highly sensitive and robust method for the quantification of specific DNA sequences based on coupling asymmetric helicase-dependent DNA amplification toelectrochemical detection is described.This method relies on the entrapment of the amplified ssDNA sequences on magnetic beads followed by a post-amplification hybridization assay to provide an added degree of specificity.Asaproof-of-concept a 84-bases long sequence specific of Mycobacterium tuberculosis is amplified at 65 °C, providing 3x10 6 amplification after 90 min.Using this system 0.5aM,corresponding to 15 copies of the target gene in 50 mL of sample,canbe successfully detected and reliably quantified under isothermal conditions in less than 4h.The assay has been applied to the detection of M. tuberculosis in sputum,pleural fluid andurine samples. Besides this application,the proposed assays is apowerful and general tool formoleculardi- agnostic that can be applied to the detection of other specific DNA sequences,taking full advantage of the plethora of genomic information now available.
URI: http://hdl.handle.net/10651/32536
ISSN: 0956-5663
Sponsored: This work has been supported by Ministerio de Economía y Competitividad (Spain)Projects CTQ2008-02429 and CTQ201231157,and the European Regional Development Fund.
Project id.: MINECO/CTQ2008-02429
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