Proof-of-Concept Nanoparticle-Based Biosensor for Detecting the African Swine Fever Virus Across Multiple Genotypes Using In Silico and In Vitro Approaches

African swine fever virus (ASFV) is a viral hemorrhagic disease with high lethality in domestic and wild swine, posing a critical threat to global food security and livestock economies. Rapid and accurate detection of ASFV is crucial for effective containment of outbreaks. This study evaluated a gold nanoparticle-based biosensor for the detection of ASFV by targeting the p72 gene using eight oligonucleotide probes. The objective was to identify optimal probes with high sensitivity and specificity, and broad genotypic coverage. Clustal Omega was used to perform multiple sequence alignments between each probe and diverse ASFV genomes. Percentage identity matrices were generated and visualized through heatmaps to assess hybridization strength across genotypes. The biosensor was then tested with synthetic ASFV DNA at a 5 min reaction time, using spectrophotometric analysis to evaluate detection. Sensitivity was measured through serial dilutions of target DNA, and specificity was confirmed using nontarget bacterial DNA. Probes 2 (40 bp, 50.0% GC content) and 5 (60 bp, 54.2% GC content) demonstrated the strongest overall performance, achieving detection of 550 copies with no cross-reactivity and strong binding across multiple ASFV genotypes. Statistical analysis using Spearman's rank correlation demonstrated that GC content was significantly associated with sensitivity (ρ = -0.80, <i>p</i> = 0.016), while probe length, secondary structure stability, and binding advantage showed no significant relationships. This study underscored the importance of integrating genomic alignment tools with experimental biosensor validation to enhance probe design.