title: Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification.
abstract: 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 colorimetrically 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°C, achieving a limit of detection of 1.3×10(-13) M (4×10(6) copies in 50 μL) for the colorimetric assay and 3.3×10(-14) M (2×10(5) copies in 10 μL) for the chronoamperometric assay. The system was demonstrated to be highly specific with negligible cross-reactivity with non-complementary targets or primers.
authors: Nanobiotechnology and Bioanalysis Group, Departament d'Enginyeria Química, Universitat Rovira I Virgili, 26 Paisos Catalans, 43007 Tarragona, Spain.
authors: Jonathan Sabaté
authors: JS
authors: del Río
authors: Nanobiotechnology and Bioanalysis Group, Departament d'Enginyeria Química, Universitat Rovira I Virgili, 26 Paisos Catalans, 43007 Tarragona, Spain.
authors: Nouran
authors: N
authors: Yehia Adly
authors: Nanobiotechnology and Bioanalysis Group, Departament d'Enginyeria Química, Universitat Rovira I Virgili, 26 Paisos Catalans, 43007 Tarragona, Spain.
authors: Josep Lluis
authors: JL
authors: Acero-Sánchez
authors: Nanobiotechnology and Bioanalysis Group, Departament d'Enginyeria Química, Universitat Rovira I Virgili, 26 Paisos Catalans, 43007 Tarragona, Spain.
authors: Olivier Y F
authors: OY
authors: Henry
authors: Nanobiotechnology and Bioanalysis Group, Departament d'Enginyeria Química, Universitat Rovira I Virgili, 26 Paisos Catalans, 43007 Tarragona, Spain; Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys, 23, 08010 Barcelona, Spain. Electronic address: ciara.osullivan@urv.cat.
authors: Ciara K
authors: CK
authors: O'Sullivan
mesh_terms: D001483:Base Sequence; D015342:DNA Probes / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004867:Equipment Design; D005604:Francisella tularensis / Q000235:genetics*; D006801:Humans; D057230:Limit of Detection; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D045522:Recombinases / Q000378:metabolism; D014406:Tularemia / Q000382:microbiology
chemical_list: D015342:DNA Probes; D004269:DNA, Bacterial; D045522:Recombinases
keywords: Francisella tularensis; Genosensor; Recombinase polymerase amplification; Solid phase PCR