title: Isothermal solid-phase amplification system for detection of Yersinia pestis.
abstract: DNA amplification is required for most molecular diagnostic applications, but conventional polymerase chain reaction (PCR) has disadvantages for field testing. Isothermal amplification techniques are being developed to respond to this problem. One of them is the recombinase polymerase amplification (RPA) that operates at isothermal conditions without sacrificing specificity and sensitivity in easy-to-use formats. In this work, RPA was used for the optical detection of solid-phase amplification of the potential biowarfare agent Yersinia pestis. Thiolated forward primers were immobilized on the surface of maleimide-activated microtitre plates for the quantitative detection of synthetic and genomic DNA, with elongation occurring only in the presence of the specific template DNA and solution phase reverse primers. Quantitative detection was achieved via the use of biotinylated reverse primers and post-amplification addition of streptavidin-HRP conjugate. The overall time of amplification and detection was less than 1 h at a constant temperature of 37 °C. Single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) sequences were detected, achieving detection limits of 4.04*10(-13) and 3.14*10(-16) M, respectively. The system demonstrated high specificity with negligible responses to non-specific targets.
authors.affiliation: Interfibio Research Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain.
authors.forename: Olena
authors.initials: O
authors.lastname: Mayboroda
authors.affiliation: Interfibio Research Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain.
authors.forename: Angel
authors.initials: A
authors.lastname: Gonzalez Benito
authors.affiliation: Interfibio Research Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Sabaté del Rio
authors.affiliation: Interfibio Research Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain.
authors.forename: Marketa
authors.initials: M
authors.lastname: Svobodova
authors.affiliation: Friedrich-Loeffler-Institut, Naumburger Str. 96a, 07743, Jena, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Julich
authors.affiliation: Friedrich-Loeffler-Institut, Naumburger Str. 96a, 07743, Jena, Germany.
authors.forename: Herbert
authors.initials: H
authors.lastname: Tomaso
authors.affiliation: Interfibio Research Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain. ckosulli@etse.urv.es.|ICREA, Passeig Lluís Companys 23, 08010, Barcelona, Spain. ckosulli@etse.urv.es.
authors.forename: Ciara K
authors.initials: CK
authors.lastname: O'Sullivan
authors.affiliation: Interfibio Research Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain. ioanis.katakis@urv.cat.
authors.forename: Ioanis
authors.initials: I
authors.lastname: Katakis
mesh_terms: D004247:DNA / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation / Q000379:methods*; D015010:Yersinia pestis / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004269:DNA, Bacterial; D004247:DNA
keywords: ELONA; Recombinase polymerase amplification; Solid-phase detection; Yersinia pestis