Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification

Solid-phase isothermal DNA amplification was performed exploiting the homology protein recombinase A (recA). The system was primarily tested on maleimide activated microtitre plates as a proof-of-concept and later translated to an electrochemical platform. In both cases, forward primer for Francisella tularensis holarctica genomic DNA was surface immobilised via a thiol or an amino moiety and then elongated during the recA mediated amplification, carried out in the presence of specific target sequence and reverse primers. The formation of the subsequent surface tethered amplicons was either color-imetrically or electrochemically monitored using a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the elongated strand. The amplification time was optimised to amplify even low amounts of DNA copies in less than an hour at a constant temperature of 37 degrees C, achieving a limit of detection of 1.3 x 10(-13) M (4 x 10(6) copies in 50 mu L) for the colorimetric assay and 3.3 x 10(-14) M (2 x 10(5) copies in 10 mu L) for the chronoamperometric assay. The system was demonstrated to be highly specific with negligible cross-reactivity with non-complementary targets or primers. (C) 2013 Elsevier B.V. All rights reserved.