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Electrochemical detection of Piscirickettsia salmonis genomic DNA from salmon samples using solid-phase recombinase polymerase amplification

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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 · 10−8 μg ml−1 (3 · 103 copies 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.

Schematic of use of electrochemical RPA for detection of Psiricketessia salmonis in salmon liver

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Authors and Affiliations

  1. Nanobiotechnology and Bioanalysis Group, Departament d’Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain

    Jonathan Sabaté del Río, Marketa Svobodova & Ciara K. O’Sullivan

  2. Centro de Investigación y Gestión de Recursos Naturales, Facultad de Ciencias, Universidad de Valparaíso, Av. Gran Bretaña 1111, 2340000, Valparaíso, Chile

    Paulina Bustos & Pablo Conejeros

  3. Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys, 23, 08010, Barcelona, Spain

    Ciara K. O’Sullivan

Authors
  1. Jonathan Sabaté del Río
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  2. Marketa Svobodova
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  3. Paulina Bustos
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  4. Pablo Conejeros
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  5. Ciara K. O’Sullivan
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Correspondence to Ciara K. O’Sullivan.

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Published in the topical collection Isothermal Nucleic Acid Amplification in Bioanalysis with guest editor Maria Jesus Lobo Castañón.

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del Río, J.S., Svobodova, M., Bustos, P. et al. Electrochemical detection of Piscirickettsia salmonis genomic DNA from salmon samples using solid-phase recombinase polymerase amplification. Anal Bioanal Chem 408, 8611–8620 (2016). https://doi.org/10.1007/s00216-016-9639-0

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  • DOI: https://doi.org/10.1007/s00216-016-9639-0

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