Toubanaki Dimitra K, Karagouni Evdokia
Laboratory of Cellular Immunology, Department of Microbiology, Hellenic Pasteur Institute, 127 Vas. Sofias Ave., 11521 Athens, Greece.
J Anal Methods Chem. 2018 Feb 20;2018:7691014. doi: 10.1155/2018/7691014. eCollection 2018.
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.
神经坏死病毒（诺达病毒）已导致全球水产养殖业大量死亡，造成了巨大的经济和环境影响。一种用于鉴定诺达病毒基因型的快速低成本检测方法，对于学术界和养鱼业的中小型实验室的疫苗和诊断应用可能具有重要意义。开发并优化了一种用于同时检测最常见诺达病毒基因型（RGNNV和SJNNV）的双侧向流动生物传感器。该双生物传感器由两个基于抗体的测试区（分别指示每种基因型）和一个对照区组成。通过用抗生物素蛋白抗体功能化的金纳米颗粒使阳性信号可视化，检测在20分钟内完成。优化研究包括确定用于测试区构建的抗体类型和数量、选择用于产生高信号的金纳米颗粒缀合物类型以及确定检测分析参数。优化后，用健康鱼样本和感染RGNNV诺达病毒的鱼样本对生物传感器进行了评估。该检测方法的成本估计不到3欧元，包括所需的试剂和生物传感器。这项工作朝着制作用于诺达病毒基因分型的双侧向流动生物传感器迈出了重要一步；在该检测方法适合用于开发诊断试剂盒之前，还需要用临床样本进行进一步评估。
