Attomolar quantification of Mycobacterium tuberculosis by asymmetric helicase dependent isothermal DNA-amplification and electrochemical detection

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.