Abstract
Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the main pathogens of grapes, causing a significant loss in yield and decrease in quality for this agricultural plant. For efficient widespread control of this infection, rapid and simple analytical techniques of on-site testing are requested as a complementary addition for the currently applied hybridization (PCR) and immunoenzyme (ELISA) approaches. The given paper presents development and approbation of the immunochromatographic assay (ICA) for rapid detection of GLRaV-3. The ICA realizes a sandwich immunoassay format with the obtaining complexes ((antibody immobilized on immunochromatographic membrane)–(virus in the sample)–(antibody immobilized on gold nanoparticles (GNP)) during sample flow along the membrane compounds of the test strip. Three preparations of GNPs were compared for detection of GLRaV-3 at different dilutions of virus-containing sample. The GNPs with maximal average diameters of 51.0 ± 7.9 nm provide GLRaV-3 detection for its maximal dilutions, being 4 times more than when using GNPs with a diameter of 28.3 ± 3.3 nm, and 8 times more than when using GNPs with a diameter of 18.5 ± 3.3 nm. Test strips have been manufactured using the largest GNPs conjugated with anti-GLRaV-3 antibodies at a ratio of 1070:1. When testing samples containing other grape wine viruses, the test strips have not demonstrated staining in the test zone, which confirms the ICA specificity. The approbation of the manufactured test strips indicated that when using ELISA as a reference method, the developed ICA is characterized by a sensitivity of 100% and a specificity of 92%. If PCR is considered as a reference method, then the sensitivity of ICA is 93% and the specificity is 92%. The proposed ICA can be implemented in one stage without the use of any additional reactants or devices. The testing results can be obtained in 10 min and detected visually. It provides significant improvement in GLRaV-3 detection, and the presented approach can be transferred for the development of test systems for other grape wine pathogens.
Highlights
Viral diseases are one of the main threats to the cultivation of grapes
The sandwich immunochromatographic assays (ICAs) with the formation of–(antigen)–(GNP-labeled antibody) complexes has been chosen for realization in this study
gold nanoparticles (GNPs)–antibody conjugate applied to the start zone (SZ) of the test strip
Summary
Viral diseases are one of the main threats to the cultivation of grapes. More than 60 viruses are pathogenic for grapes [1,2,3]. Additional agricultural risks result from grapes being a vegetatively propagated perennial crop in which pathogens accumulate while the plants grow in vineyards. Grapevine leafroll disease (GLD) is considered as one of the most common and economically. Biosensors 2018, 8, 111 significant viral diseases of this plant, which is caused by viruses of the Closteroviridae family [4,5]. Grapevine leafroll-associated virus 3 (GLRaV-3), a member of the genus Ampelovirus, is the infectious agent associated with GLD. Particles of GLRaV-3 virus are filamentous structures with a length of 1800 nm and a diameter of 12 nm with a spiral arrangement of repetitive protein subunits [4]
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