Integrated laboratory- and field-immunodiagnostics for detection of apple necrotic mosaic virus

Apple necrotic mosaic virus (ApNMV) has emerged as a major viral threat to apple production worldwide, yet its detection currently relies on molecular assays that are unsuitable for large-scale or field-level surveillance. There are no in field detection assays available for detection of this emerging virus. In this study, we report the first indigenous serology-based immunodiagnostic platforms developed specifically for ApNMV, enabling both laboratory and on-site detection. Polyclonal antibodies were generated against the recombinant ApNMV coat protein and employed to establish two complementary diagnostic formats: an indirect enzyme-linked immunosorbent assay (ELISA) for high-throughput laboratory screening and a gold nanoparticle-based lateral flow immunoassay (LFIA) for rapid field detection. The ELISA successfully detected ApNMV beyond the early growing season, supporting its utility for extended surveillance. The LFIA produced visually interpretable results within 10 minutes, employed a simple tube-based sample preparation suitable for field use, and showed no cross-reactivity with closely related apple viruses, including apple mosaic virus and apple stem grooving virus. Evaluation using field-collected apple samples demonstrated complete concordance between LFIA and RT-PCR results within the tested set, confirming its reliability for rapid detection. Collectively, this work establishes the first field-deployable immunodiagnostic system for ApNMV and provides a transferable framework for surveillance, nursery certification, and management of emerging viral diseases in perennial fruit crops.