Specific detection of virus strains by affinity-based bioassays is often limited by the availability of ligands able to differentiate among close homologues of virus coat proteins. As viruses are prone to mutation, the ligand generation should, in addition, be fast enough to allow rapid identification of new varieties. These two criteria are difficult to be fulfilled by antibodies; however, they open up opportunities for aptamer-based detection. Here we report on the feasibility of selectively detecting the apple stem pitting virus (ASPV) coat proteins (PSA-H, MT32) using original DNA aptamers. Surface plasmon resonance (SPR) imaging was used together with aptamer-modified sensor chips to optimize the aptamer immobilization for highest sensitivity and to characterize the aptamer-virus coat protein binding. Different parameters affecting this binding, such as the aptamer flanking, surface coverage, and type of spacer molecules, were identified and their influence was determined. A direct label-free method is proposed for assessing the ASPV based on the detection of the respective virus coat proteins in plant extracts.

Although the significance of molecular diagnostics in routine plant virus detection is rapidly growing, the preferred methods are still antibody-based enzyme immunoassays. In the past decade, aptamers have been demonstrated to be viable alternatives of antibodies in many applications. We set out to select apple stem pitting virus (ASPV)-specific aptamers and to apply them as antibody substitutes in various immunoassay methods. The applied systematic evolution of ligands by exponential enrichment (SELEX) procedure resulted in highly discriminative aptamers selectively binding to the target virus coat protein even in complex protein matrixes. We developed protocols for exploitation of aptamers in diverse plant virus diagnosis methods, such as dot and Western blot analyses and enzyme-linked oligonucleotide assay (ELONA). Our selected aptamers proved to be superior to the available antibody in all aspects. In contrast to the antibody, the aptamers decorate both native and denaturated proteins, and ELONA produces higher signal intensity than traditional enzyme-linked immunosorbent assay (ELISA) with virus-infected plant extract. Summarily, our results present the selection and practical utilization of first plant virus-specific aptamers. Most important, the first application of ELONA for virus detection is demonstrated, which proposes a novel, more flexible, and cost-effective means of virus diagnostics.

We introduce Amplified Luminescent Proximity Homogenous Assay (ALPHA) to assess the K(D) value of aptamer-protein complexes as demonstrated through the study of apple stem pitting virus coat protein-specific aptamers. This method can be used as a simple, cost-effective method for screening aptamer-target protein interactions during aptamer selection.