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Analytical and Bioanalytical Chemistry

, Volume 408, Issue 30, pp 8611–8620 | Cite as

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

  • Jonathan Sabaté del Río
  • Marketa Svobodova
  • Paulina Bustos
  • Pablo Conejeros
  • Ciara K. O’Sullivan
Research Paper
Part of the following topical collections:
  1. Isothermal Nucleic Acid Amplification in Bioanalysis

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.

Graphical abstract

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

Keywords

Electrochemical genosensor DNA Recombinase polymerase amplification (RPA) Piscirickettsia salmonis 

Notes

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Supplementary material

216_2016_9639_MOESM1_ESM.pdf (50 kb)
ESM 1 (PDF 49 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Jonathan Sabaté del Río
    • 1
  • Marketa Svobodova
    • 1
  • Paulina Bustos
    • 2
  • Pablo Conejeros
    • 2
  • Ciara K. O’Sullivan
    • 1
    • 3
  1. 1.Nanobiotechnology and Bioanalysis Group, Departament d’Enginyeria QuímicaUniversitat Rovira i VirgiliTarragonaSpain
  2. 2.Centro de Investigación y Gestión de Recursos Naturales, Facultad de CienciasUniversidad de ValparaísoValparaísoChile
  3. 3.Institució Catalana de Recerca i Estudis AvançatsBarcelonaSpain

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