Development of a Nanoparticle-based Lateral Flow Strip Biosensor for Visual Detection of Whole Nervous Necrosis Virus Particles

Abstract

Effective analysis of pathogens causing human and veterinary diseases demands rapid, specific and sensitive detection methods which can be applied in research laboratory setups and in field for routine diagnosis. Paper lateral flow biosensors (LFBs) have been established as attractive tools for such analytical applications. In the present study a prototype LFB was designed for whole particles (virions) detection of nodavirus or fish nervous necrosis virus. Nodavirus is an important threat in the aquaculture industry, causing severe economic losses and environmental problems. The LFB was based on polyclonal antibodies conjugated on gold nanoparticles for signal visualization. Brain and retinas from fish samples were homogenized, centrifuged and the supernatant was directly applied on the LFB. formation of a red test line was indicative of nodavirus virions presence. nodavirus visual detection was completed in short time (30 min). Key factors of the LFB development influencing the assays' detection limit were characterized and the optimum parameters were determined, enabling increased efficiency, excluding non-specific interactions. Therefore, the proposed LFB assay consists a robust, simple, low cost and accurate method for detection of nodavirus virions in fish samples. The proposed biosensor is ideal for development of a commercial kit to be used on aquaculture facilities by fish farmers. It is anticipated that disease monitoring and environmental safety will benefit from the simplification of time consuming and costly procedures.Aquaculture is essential to cover fish-product demands, providing seafood in high quantities, and covering more than the half amount of fish consumed worldwide. This fact drives a strong demand for high production efficiency in aquaculture industry in order to cover the feeding needs of the world's growing population, in the middle of an increasing environmental crisis 1,2 . As a result, the aquaculture industry has continuously increased profits in a high rate. However, outbreaks of diseases caused by infectious agents are significantly restricting intensified aquaculture. According to literature 3 , 22.6% of all disease outbreaks are caused by viruses. Among these, viral nervous necrosis (VNN), also named vacuolating encephalopathy and retinopathy or encephalomyelitis, is a devastating disease, which induces cell necrosis accompanied by vacuolation in fish retina and brain. Its clinical symptoms include changes in skin color with abnormal swimming, low feed ingestion and altered buoyancy in affected fish. The disease is caused by nervous necrosis virus (NNV) or nodavirus, affecting more than 30 different fish species, worldwide. VNN causes high mortalities (80-100% in several species e.g. European sea bass), emerging as a major problem especially in the Mediterranean area, since it cannot be prevented by vaccination or effective treatment 4-6 .Fish nervous necrosis virus (belonging to Betanodavirus genus and Nodaviridae family) is icosahedral, and non-enveloped (∼25 nm in diameter). I...

Development and evaluation of colloidal gold immunochromatography test strip for rapid diagnosis of nervous necrosis virus in golden grey mullet (Chelon aurata)

“…As shown in Figure 4a, the colour intensity of the test line of the strip decreased depending on the serial dilution of the virus and the last dilution that the strip test gave a positive result was 10 3 TCID 50 /ml. The present assay showed comparable sensitivity to previously reported methods for virus detection such as ELISA (2–3 × 10 3 TCID 50 ) and lateral flow strip biosensor (6 × 10 –3 TCID 50 /0.5 μl; Fenner et al., 2006; Toubanaki et al., 2020). To our knowledge, there is only one publication dealing with applying an immunochromatography‐based lateral flow strip assay for the detection of NNV using the RGNNV‐specific monoclonal antibodies with a detection limit of 10 5.05 TCID 50 /100 μl (Shyama et al., 2020).…”

“…The use of lateral flow assays (LFA), also known as the immunochromatography assays or strip tests, has recently attracted attention as a new method for rapidly identifying pathogens in infected fish within a fish‐farm setting (Liu et al., 2017; Shyama et al., 2020; Toubanaki et al., 2020). Compared to traditional immunoassays, LFA is a simple, user‐friendly, one‐step assay, with no wash steps, providing results within minutes, with only a small sample volume required.…”

Principles and Recent Advances in Biosensors for Pathogens Detection

“…The structure of the antibody consists of two similar copies of small and big polypeptide chains that are connected by non-covalent and disulfide bonds to build a Yshaped biomolecule of 10 nm. [16] The small and heavy chains include variable and constant regions in which each region plays a substantial functional role in the antibody. The variable region is important for binding specifically to the antigen, whereas the constant region is accountable for the associations with different molecules.…”

Disturbance of cellular homeostasis as a molecular risk evaluation of human endothelial cells exposed to nanoparticles

“…Sterilizing filtration was used for NPs sized < 220 nm, whereas for particles with greater sizes, NPs were lyophilized and then resuspended in sterile water. The final step referred to the analyzed NP description of entrapment efficacy (% of encapsulated drug) calculation described previously [66][67][68] .…”