A portable one-tube ERA-CRISPR/Cas12a assay for rapid detection of African swine fever virus

African swine fever virus (ASFV) poses a serious threat to the global swine industry, creating an urgent need for rapid, sensitive, and field-deployable diagnostic methods. This study developed a portable one-tube detection platform that integrates enzymatic recombinase amplification (ERA) with CRISPR/Cas12a-mediated fluorescence detection. A customized dual-layer reaction tube was designed to physically separate ERA amplification from the CRISPR/Cas12a reaction before detection, thereby reducing direct interaction between ERA amplification and CRISPR/Cas12a detection during the initial amplification stage. Three forward primers, three reverse primers, and a crRNA targeting the conserved ASFV B646L gene were designed and screened to establish the optimal assay. Under optimized conditions, the assay specifically detected ASFV DNA without cross-reactivity with common swine pathogens, including PEDV, PRRSV, PRV, SVA, CSFV, and PCV2. The analytical limit of detection was 1.21 × 10 1 copies/μL for the plasmid standard, and the assay showed good repeatability and reproducibility, with coefficients of variation below 6.9% and 6.75%, respectively. In simulated clinical serum samples, all positive and negative samples were correctly identified. Fluorescence signals were monitored using a smartphone-connected portable thermostatic nucleic acid amplification analyzer. These findings indicate that the one-tube ERA-CRISPR/Cas12a platform provides a rapid, portable, and contamination-resistant approach for potential point-of-care ASFV screening.