title: Enhanced solid-phase recombinase polymerase amplification and electrochemical detection.
abstract: Recombinase polymerase amplification (RPA) is an elegant method for the rapid, isothermal amplification of nucleic acids. Here, we elucidate the optimal surface chemistry for rapid and efficient solid-phase RPA, which was fine-tuned in order to obtain a maximum signal-to-noise ratio, defining the optimal DNA probe density, probe-to-lateral spacer ratio (1:0, 1:1, 1:10 and 1:100) and length of a vertical spacer of the probe as well as investigating the effect of different types of lateral spacers. The use of different labelling strategies was also examined in order to reduce the number of steps required for the analysis, using biotin or horseradish peroxidase-labelled reverse primers. Optimisation of the amplification temperature used and the use of surface blocking agents were also pursued. The combination of these changes facilitated a significantly more rapid amplification and detection protocol, with a lowered limit of detection (LOD) of 1 · 10-15M. The optimised protocol was applied to the detection of Francisella tularensis in real samples from hares and a clear correlation with PCR and qPCR results observed and the solid-phase RPA demonstrated to be capable of detecting 500 fM target DNA in real samples. Graphical abstract Relative size of thiolated lateral spacers tested versus the primer and the uvsx recombinase protein.
authors.affiliation: Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain.
authors.forename: Jonathan Sabaté
authors.initials: JS
authors.lastname: Del Río
authors.affiliation: Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain.
authors.forename: Ivan Magriñà
authors.initials: IM
authors.lastname: Lobato
authors.affiliation: Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain.
authors.forename: Olena
authors.initials: O
authors.lastname: Mayboroda
authors.affiliation: Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain.
authors.forename: Ioanis
authors.initials: I
authors.lastname: Katakis
authors.affiliation: Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain. ciara.osullivan@urv.cat.|Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys, 23, 08010, Barcelona, Spain. ciara.osullivan@urv.cat.
authors.forename: Ciara K
authors.initials: CK
authors.lastname: O'Sullivan
mesh_terms: D000818:Animals; D015342:DNA Probes / Q000032:analysis / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D055664:Electrochemical Techniques / Q000379:methods*; D005604:Francisella tularensis / Q000235:genetics* / Q000302:isolation & purification; D057230:Limit of Detection; D008958:Models, Molecular; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011817:Rabbits; D045522:Recombinases / Q000737:chemistry; D059629:Signal-To-Noise Ratio; D014406:Tularemia / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary
chemical_list: D015342:DNA Probes; D004269:DNA, Bacterial; D045522:Recombinases
keywords: Electrochemical genosensor; Real samples from hares; Solid-phase recombinase polymerase amplification; Surface chemistry