Rapid point-of-care lateral flow assay for differentiation of capripoxviruses using RPA-CRISPR-Cas12a.

The Capripoxviruses (CaPVs) are highly contagious viruses that includes sheeppox virus, goatpox virus, and lumpy skin disease virus infecting sheep, goats, and cattle respectively resulting in significant morbidity and mortality. This study presents a diagnostic tool based on recombinase polymerase amplification (RPA) assisted with CRISPR-Cas12a platform coupled with a lateral flow assay (LFA) that facilitate timely detection and differentiation of Capripox viruses, and support effective surveillance programs. The assay operates through the cleavage of a dual labelled oligonucleotide reporter molecule (Biotin-Fam), generating a detectable signal. The results can be interpreted either using a fluorescence-based detection system or through a lateral flow assay (LFA), where gold nanoparticles conjugated with anti-FAM antibodies enable visual signal detection. Under optimized assay conditions (50 nM LbCas12a and 1 µM reporter probe with a reaction time of 25 min), the CRISPR-Cas12a platform along with RPA (39°C for 20 min) substantially enhanced analytical sensitivity, enabling reliable detection of as few as 10 copies/µL of viral DNA. The specificity of the assay was rigorously evaluated using individual targets, mixed-template samples, and 90 clinical specimens, demonstrating complete species-specific discrimination without any detectable cross-reactivity. Collectively, the developed single-gene RPA-CRISPR-Cas12a lateral flow assay (LFA) represents a rapid, highly sensitive, and specific point-of-care diagnostic platform, providing visual readout by the naked eye and offering significant potential for decentralized veterinary diagnostics and field-based disease surveillance.