title: Electrochemical detection of Piscirickettsia salmonis genomic DNA from salmon samples using solid-phase recombinase polymerase amplification.
abstract: Electrochemical detection of solid-phase isothermal recombinase polymerase amplification (RPA) of Piscirickettsia salmonis in salmon genomic DNA is reported. The electrochemical biosensor was constructed by surface functionalization of gold electrodes with a thiolated forward primer specific to the genomic region of interest. Solid-phase RPA and primer elongation were achieved in the presence of the specific target sequence and biotinylated reverse primers. The formation of the subsequent surface-tethered duplex amplicons was electrochemically monitored via addition of streptavidin-linked HRP upon completion of solid-phase RPA. Successful quantitative amplification and detection were achieved in less than 1 h at 37 °C, calibrating with PCR-amplified genomic DNA standards and achieving a limit of detection of 5 · 10-8μg ml-1(3 · 103copies in 10 μl). The presented system was applied to the analysis of eight real salmon samples, and the method was also compared to qPCR analysis, observing an excellent degree of correlation. Graphical abstract Schematic of use of electrochemical RPA for detection of Psiricketessia salmonis in salmon liver.
authors.affiliation: Nanobiotechnology and Bioanalysis Group, 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: Nanobiotechnology and Bioanalysis Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain.
authors.forename: Marketa
authors.initials: M
authors.lastname: Svobodova
authors.affiliation: Centro de Investigación y Gestión de Recursos Naturales, Facultad de Ciencias, Universidad de Valparaíso, Av. Gran Bretaña 1111, 2340000, Valparaíso, Chile.
authors.forename: Paulina
authors.initials: P
authors.lastname: Bustos
authors.affiliation: Centro de Investigación y Gestión de Recursos Naturales, Facultad de Ciencias, Universidad de Valparaíso, Av. Gran Bretaña 1111, 2340000, Valparaíso, Chile.
authors.forename: Pablo
authors.initials: P
authors.lastname: Conejeros
authors.affiliation: Nanobiotechnology and Bioanalysis Group, 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; D015374:Biosensing Techniques / Q000295:instrumentation; D019921:Biotinylation; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis*; D055664:Electrochemical Techniques; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry; D005393:Fish Diseases / Q000175:diagnosis* / Q000382:microbiology; D006046:Gold / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques; D053522:Piscirickettsia / Q000235:genetics* / Q000302:isolation & purification; D044225:Piscirickettsiaceae Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D045522:Recombinases / Q000737:chemistry; D012474:Salmon / Q000382:microbiology
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D004800:Enzymes, Immobilized; D045522:Recombinases; D006046:Gold; D006735:Horseradish Peroxidase
keywords: DNA; Electrochemical genosensor; Piscirickettsia salmonis; Recombinase polymerase amplification (RPA)