Fast and simplified CRISPR–Cas13d assay for sensitive detection of porcine deltacoronavirus

Porcine deltacoronavirus (PDCoV) poses a serious threat to swine health and causes significant economic losses in the pig industry, underscoring the need for rapid, sensitive, and field-deployable diagnostic technologies. Here, we present RAPID (Rapid Automated Portable Integrated Detection), a highly sensitive and specific nucleic acid detection platform that integrates RPA amplification with EsCas13d-based detection in a single reaction. The workflow begins with a two-minute room-temperature lysis step that releases viral RNA directly from unextracted samples, followed by a streamlined one-tube reaction completed within 30 minutes. Optimization of the reaction buffer, reverse transcription, primer design, and crRNA selection has enabled efficient single-step amplification and detection, minimizing assay complexity and the risk of contamination. The incorporation of lyophilization technology provides long-term reagent stability and allows for transport under ambient conditions, thus eliminating reliance on cold-chain logistics. Importantly, RAPID can operate effectively at both physiological temperature (37 °C) and room temperature (25 °C), removing the need for specialized equipment. Detection results can be visualized through in-tube fluorescence or lateral flow assays, providing flexible readout options. Clinical validation using porcine samples demonstrated complete concordance with RT-qPCR, achieving 100% sensitivity and specificity. With its speed, accuracy, reagent stability, and instrument independence, RAPID offers a practical and affordable POCT solution, particularly suited for on-farm surveillance and deployment in resource-limited settings.