DAS-ELISA-based detection and RT-PCR validation for precise and timely analysis of Citrus tristeza virus infection in Nepalese citrus species

Citrus viral infections pose a substantial threat to the global citrus industry, resulting in multitudinous economic losses annually. Timely and precise identification of citrus viruses offers a promising avenue for proactive insights preemptively before the instigation of extensive infections. The present study was devised to expedite the prompt identification of the presence of Citrus tristeza virus (CTV) within Nepalese citrus species utilizing a Double Antibody Sandwich-Enzyme-Linked Immunosorbent Assay (DAS-ELISA), followed by its validation using reverse transcription PCR (RT-PCR). Samplings extracted from plants harvested from 74 out of the 77 administrative districts within Nepal provide substantial evidence of extensive propagation of viral contagion among citrus cultivars across 64 districts (86.48%) of the nation. A comprehensive analysis of the samples obtained from diverse geographical localities revealed that 36 districts (48.64%) exhibit mild infestation, while 9 districts (12.16%) evince moderate infestation. Moreover, samples harvested from 19 districts (25.67%) were highly infected; intriguingly, specimens from 10 districts (13.51%) manifest an absence of viral intrusion. The results have been validated with RT-PCR assay. Given the profound significance of citrus as a pivotal repository of vitamin C and its essential role in conferring advantageous health attributes to humankind, alongside its substantial contribution to the economic paradigm, a meticulous examination is imperative to safeguard these plants against an array of viral infections. Collectively, the DAS-ELISA methodology along with RT-PCR approaches constitutes an exquisitely sensitive and meticulously specific modality, poised to accelerate the expeditious detection of citrus-associated viruses within burgeoning economies like Nepal. This advancement supports timely and precise detection and diagnosis of agriculturally virulent conditions, thereby forestalling the eruption of viral epidemics and concomitantly curbing their financial repercussions.