Direct Amperometric Sensing of Fish Nodavirus RNA Using Gold Nanoparticle/DNA-Based Bioconjugates

Abstract

We describe the design of a simple and highly sensitive electrochemical bioanalytical method enabling the direct detection of a conserved RNA region within the capsid protein gene of a fish nodavirus, making use of nanostructured disposable electrodes. To achieve this goal, we select a conserved region within the nodavirus RNA2 segment to design a DNA probe that is tethered to the surface of nanostructured disposable screen-printed electrodes. In a proof-of-principle test, a synthetic RNA sequence is detected based on competitive hybridization between two oligonucleotides (biotinylated reporter DNA and target RNA) complimentary to a thiolated DNA capture probe. The method is further validated using extracted RNA samples obtained from healthy carrier Sparus aurata and clinically infected Dicentrarchus labrax fish specimens. In parallel, the sensitivity of the newly described biosensor is compared with a new real-time RT-PCR protocol. The current differences measured in the negative control and in presence of each concentration of target RNA are used to determine the dynamic range of the assay. We obtain a linear response (R2 = 0.995) over a range of RNA concentrations from 0.1 to 25 pM with a detection limit of 20 fM. The results are in good agreement with the results found by the RT-qPCR. This method provides a promising approach toward a more effective diagnosis and risk assessment of viral diseases in aquaculture.

Keywords

• betanodavirus
• amperometry
• nanosensors
• RT-PCR
• fish
• diagnosis