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.
Highlights
Among the most reported viral infections in marine aquaculture, viral encephalopathy and retinopathy (VER— known as viral nervous necrosis, VNN) caused by betanodaviruses is one of the most highly impactful pathogens in hatcheries and grow-out fish facilities
Based on phylogenetic analysis of the RNA2 segments (T4 variable region), betanodavirus isolates have been classified into four genotypes, currently accepted by the International Committee on Taxonomy of Viruses (ICTV): striped jack nervous necrosis virus (SJNNV), tiger puffer nervous necrosis virus (TPNNV), barfin flounder nervous necrosis virus (BFNNV) and redspotted grouper nervous necrosis virus (RGNNV) [3,4]
The RNA2 5 -6FAM/BHQ1-3 probe from positions 381 to 399, with a very high nucleotide similarity percentage, was designed. This fragment was present in the secondary structure of the RNA2 as the end of a branch with a very small, conserved hairpin (Figure 1C)
Summary
Among the most reported viral infections in marine aquaculture, viral encephalopathy and retinopathy (VER— known as viral nervous necrosis, VNN) caused by betanodaviruses (piscine nodavirus) is one of the most highly impactful pathogens in hatcheries and grow-out fish facilities. Based on phylogenetic analysis of the RNA2 segments (T4 variable region), betanodavirus isolates have been classified into four genotypes, currently accepted by the International Committee on Taxonomy of Viruses (ICTV): striped jack nervous necrosis virus (SJNNV), tiger puffer nervous necrosis virus (TPNNV), barfin flounder nervous necrosis virus (BFNNV) and redspotted grouper nervous necrosis virus (RGNNV) [3,4]. Until 2019, only outbreaks in sea bass species had been registered in the Tunisian and North African fishing industry, caused by the redspotted grouper virus RGNNV genotype [7,8,9]; new RGNNV/SJNNV reassortants have been recently introduced and are causing severe economic loss in sea bream species, previously considered a healthy carrier species (Chérif et al, personal communication)
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