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: RT-PCR; amperometry; betanodavirus; diagnosis; fish; nanosensors.

Figure 1

(A): Multiple sequence alignment of betanodavirus RNA2 nucleic sequences. GenBank accession numbers are listed. RNA2 probe hybridizes to positions 381–399 of the RGNNV RNA2 sequence (GenBank accession number: FJ789784). (B): 2D/3D structure of RNA2 generated by RIBOSUM-like similarity scoring. (C): Secondary structure of the RNA as end of branch with a very small, conserved hairpin.

Figure 2

Schematic illustration of the competition-based method for the detection of the viral RNA. Steps: (a) gold electrochemical deposition, (b) bioconjugation with the thiolated DNA capture probe, (c) addition of the biotinylated reporter probe and (d) addition of the sample containing the viral RNA and the thiolated reporter probe. A higher current is observed in case (c), where all the capture probes are solely hybridized with the biotinylated reporter probe, while in case (d) the competition results in a lower current. The current difference is proportional to the RNA_target concentration.

Figure 3

Amperometric measurements with synthetic RNA target at different concentrations. Inset: Linear portion of the curve between 100 fM and 25 pM.

Figure 4

Amperometric responses measured with the nodavirus-developed biosensors for 100 ng raw RNA_target extracted from two real samples (AS and SS) as compared to the negative control with no target (NTC). Inset: Linear portion of the curve by standard addition method to the real sample.

Figure 5

(A): Nodavirus isolation and characterization on SNN-1 cell line showing strong CPE seven days post-inoculation with a nodavirus positive sample incubated at 25 °C. (B): Analytical sensitivity of the real-time TaqMan PCR: Amplification curves obtained by testing tenfold serial dilutions of quantitated RNA standards. The x-axis shows the number of PCR cycles. The y-axis shows the fluorescence values on a logarithmic scale. (C): Standard curve generated from the above data. The x-axis indicates the logarithm of the RNA concentration expressed in copy number. The y-axis indicates the Ct value.