CRISPR-Cas12a–based lateral flow detection of white spot syndrome virus: a dual-target approach for detection of early and latent infection

White spot syndrome virus (WSSV; family <i>Nimaviridae</i>; taxon species <i>White spot syndrome virus</i>) is a major viral pathogen that poses a significant threat to the global shrimp industry, with early detection being the most effective strategy for disease control. We developed a CRISPR-Cas12a-based dual-target detection assay for WSSV, specifically targeting the <i>VP28</i> gene (gene product is a major envelope protein) and <i>WSSV366</i> (a latency-associated gene), optimized using Indian WSSV isolates. Our CRISPR RNAs for both targets had high efficiency, and we evaluated the assay using fluorescence-based and lateral flow strip (LFS) endpoint detection. In fluorescence assays, the Cr-WSSV assay (without recombinase polymerase amplification, RPA) detected WSSV at 3 × 10⁵ copies/μL; RPA integration significantly enhanced sensitivity, allowing detection at as low as 20 and 200 copies for <i>VP28</i> and <i>WSSV366</i>, respectively, with 100% specificity. We developed a CRISPR-based LFS assay with optimized FAM-biotin reporter concentrations of 100 nM and 250 nM, yielding robust and reproducible results for improved field applicability. Performance evaluation confirmed lack of cross-reactivity to other WOAH-listed shrimp pathogens, while maintaining detection limits of 20 and 200 copies of <i>VP28</i> and <i>WSSV366</i>. Clinical validation further demonstrated that the RPA-Cr-WSSV-LFS assay successfully detected <i>WSSV366</i> even in <i>VP28</i>-negative samples, underscoring the importance of detecting <i>WSSV366</i> in latent infections. Our rapid, cost-effective, and highly sensitive CRISPR-Cas-based assay enhances WSSV surveillance and biosecurity in shrimp aquaculture by incorporating structural and latency-associated gene markers, making it a promising alternative to conventional molecular testing.