Towards a Dual Lateral Flow Nanobiosensor for Simultaneous Detection of Virus Genotype-Specific PCR Products

Abstract

Nervous necrosis virus (nodavirus) has been responsible for mass mortalities in aquaculture industry worldwide, with great economic and environmental impact. A rapid low-cost test to identify nodavirus genotype could have important benefits for vaccine and diagnostic applications in small- and medium-scale laboratories in both academia and fish farming industry. A dual lateral flow biosensor for simultaneous detection of the most prevalent nodavirus genotypes (RGNNV and SJNNV) was developed and optimized. The dual biosensor consisted of two antibody-based test zones, indicative of each genotype, and a control zone. The positive signals were visualized by gold nanoparticles functionalized with anti-biotin antibody, and the detection was completed within 20 min. Optimization studies included antibody type and amount determination for test zone construction, gold nanoparticle conjugate type selection for high signal generation, and detection assay parameter determination. Following optimization, the biosensor was evaluated with healthy and RGNNV-nodavirus-infected fish samples. The proposed assay's cost was estimated to be less than 3 €, including the required reagents and biosensor. This work presents important steps towards making a dual lateral flow biosensor for nodavirus genotyping; further evaluation with clinical samples is needed before the test is appropriate for diagnostic kit development.

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

“…More rapid detection methods have also been developed for virus detection, including microfluidic chips based on RT‐PCR (Kuo et al., 2012), reverse transcription loop‐mediated isothermal amplification (RT‐LAMP; Suebsing et al., 2013) and RT‐LAMP with lateral flow dipstick (RT‐LAMP‐LFD; Lin et al., 2014). More recently, nucleic acids lateral flow biosensors were developed for detection of NNV in European sea bass; however, these assays require a sample pretreatment step for isolation and/or amplification of the target analyte (Toubanaki & Karagouni, 2018; Toubanaki et al., 2015). Although the methods described above are effective and can be used to accurately detect NNV, they are unsuitable for on‐site detection in fish farms because they require specialized equipment and reagents, skilled operators and multistep sample preparation.…”