ArticleArchives of virology2026
A field deployable duplex RPA-CRISPR/Cas12a assay for rapid and sensitive detection of African swine fever virus.
Article in Archives of virology, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
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.
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