Here, weestablish sCRAM (suboptimal protospacer adjacent motif (PAM)-mediated CRISPR RNA and Cas12a nuclease combined with multienzyme isothermal rapid amplification), a rapid and extraction-free diagnostic platform that detects ASFV within 20 min. sCRAM integrates a rapid nucleic acid release step (40℃, 5 min) with a one-tube reaction combining multienzyme isothermal amplification (MIRA) and a suboptimal PAM-mediated CRISPR/
Cas12a system (37℃, 15 min), enabling instrument-free visual readout via UV light or lateral flow strips (LFS).sCRAM demonstrates single-copy detection sensitivity and exhibits no cross-reactivity with five common swine pathogens (PRRSV, PPV, JEV, PCV, and PRV). The evaluation of 111 simulated and clinical samples (including blood, plasma, and swabs) revealed a 100 % concordance with qPCR for UV readout and 98.20 % for LFS
readout, with both methods exhibiting 100 % specificity. The simplicity, speed, and field adaptability of sCRAM underscore its potential for decentralized ASFV surveillance in resource-limited settings, offering a powerful tool for outbreak containment.
In this study, we developed sCRAM, a rapid and field-deployable ASFV detection platform integrating suboptimal PAM-mediated CRISPR/Cas12a with multienzyme isothermal amplification. The assay achieves single-copy 1 copy/uL sensitivity within 20 min and shows no cross-reactivity with major swine pathogens. Clinical validation across blood, plasma, and swab samples demonstrated full concordance with qPCR using UV light readout and 98.20 % accuracy via lateral flow strips. With simplified operation, high sensitivity and specificity, and visual result interpretation, sCRAM offers strong potential for on-site ASFV surveillance and broader point-of-care diagnostic applications.
