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.
Highlights
Aquaculture is essential to cover fish-product demands, providing seafood in high quantities, and covering more than the half amount of fish consumed worldwide
Eagle Minimum Essential Medium (EMEM), complete and incomplete Freund’s adjuvant (CFA and IFA, respectively), anti-rabbit IgG-horseradish peroxidase (HRP), poly-L-lysine, anti-rabbit IgG antibodies, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate, Au NPs stabilized in phosphate-buffered saline (PBS) (5, 10, 20, 30, 60 nm), or citrate buffer (40 nm), siliconized tubes and Tween-20 were obtained from Sigma-Aldrich (Steinhem, Germany)
The biosensor is dipped in a developing solution, which is transferred through the lateral flow biosensors (LFBs) by capillary action
Summary
Aquaculture is essential to cover fish-product demands, providing seafood in high quantities, and covering more than the half amount of fish consumed worldwide. The disease is caused by nervous necrosis virus (NNV) or nodavirus, affecting more than 30 different fish species, worldwide. Other approaches rely on immunomagnetic reduction, magnetic beads conjugated to sequence-specific captured probes or molecular beacons, and gold nanoparticles based nucleic acid lateral flow biosensors, summarized in[8,9] Despite their advantages, the mentioned methods have drawbacks that restrict their use on site. ELISA-based methods use expensive kits, including labelled antibodies which are combined with cumbersome equipment[10,11] All these demands limit their wide use for routine testing, especially in poor-equipped laboratories, as well as their accessibility and usability in field diagnosis. Colloidal gold has been widely used because of its excellent optical and chemical stability[16,17]
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