A field deployable duplex RPA-CRISPR/Cas12a assay for rapid and sensitive detection of African swine fever virus.

African swine fever (ASF) is a notifiable transboundary disease of domestic pigs and wild suids that causes severe haemorrhagic fever, with high mortality, and substantial socio-economic losses worldwide. In the absence of effective vaccines or therapeutics, ASF control relies primarily on early diagnosis, strict biosecurity, and stamping-out strategies. In this study, we developed a rapid, field-deployable recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay for the detection of African swine fever virus using both fluorescence- and lateral flow strip (LFS)-based readouts. The assay operates isothermally at 37 °ºC and enables rapid, equipment-free visual detection suitable for on-site testing. To improve diagnostic sensitivity and robustness, a dual-target approach was employed targeting the highly conserved C-terminal region of the p72 (B646L) gene and the early-expressing p22 (KP177R) gene. In the uniplex format, fluorescence-based assays achieved limits of detection of 10.18 and 13.68 copies/reaction for p72 and KP177R, respectively, whereas the LFS format detected 1,018 and 1,368 copies/reaction. Notably, the duplex RPA-CRISPR/Cas12a assay demonstrated enhanced sensitivity with detection limits of 1.02 copies/reaction in the fluorescence format and 102 copies/reaction in the LFS format. The assay showed 100% analytical specificity against other porcine viral pathogens and strong agreement with real-time PCR. Furthermore, lyophilized reagents formulated in a two-tube format retained their analytical performance after storage at 4 °C for one week, highlighting the platform's potential for decentralized ASF surveillance and outbreak response.