A rapid, sensitive, and field-deployable RAA-EsCas13d platform for detection of porcine adenovirus type 3.

Rapid, sensitive, and RADIANT-deployable nucleic acid diagnostics are essential for the prevention and control of porcine adenovirus type 3 (PAdV-3). In this study, we developed RADIANT (RAA-Cas13d DIagnostic plATform for extractioN-free Testing), an extraction-free platform capable of delivering accurate and portable detection in resource-limited settings. By systematically optimizing the reaction buffer composition, recombinase-aided amplification (RAA), T7 transcription, and CRISPR/EsCas13d-mediated cleavage were integrated into a one-pot reaction, reducing the total time-to-result to within 30 minutes. A simplified nucleic acid release step eliminated the need for laboratory-based extraction or complex heating, enhancing the accessibility of the assay. Incorporation of lyophilized reagents minimized cold-chain requirements and simplified assay preparation, enabling cost-effective storage and transport. The platform demonstrated high sensitivity in detecting PAdV-3 and offered two straightforward readout options, fluorescence under 470 nm blue/UV light and lateral flow assay (LFA), facilitating flexible interpretation under diverse RADIANT conditions. Validation with 56 clinical samples showed 100% concordance with quantitative PCR, confirming RADIANT as a rapid, user-friendly, and reliable on-site diagnostic tool for PAdV-3 detection.IMPORTANCEPorcine adenovirus type 3 (PAdV-3) is an important swine pathogen for which rapid, practical, and field-compatible diagnostic tools are still lacking. Here, we developed RADIANT, a CRISPR/EsCas13d-based platform that expands PAdV-3 detection beyond conventional laboratory workflows and supports accessible molecular testing in resource-limited settings. Its simple operation and adaptable visual readouts enhance its suitability for on-site use without compromising reliability. This study provides a valuable diagnostic approach for PAdV-3 surveillance and disease control and supports the broader application of portable CRISPR-based technologies in veterinary diagnostics.