Development and Preliminary Analytical Evaluation of Real-Time PCR Assays for African Swine Fever Virus Detection

African swine fever virus (ASFV) is a large, double-stranded DNA virus that poses a major threat to the pig industry, highlighting the need for reliable molecular diagnostics. This study reports on the design and analytical development of two primer–probe systems targeting conserved ASFV genomic regions (B646L/p72), supported by in silico assessment and laboratory testing with synthetic DNA constructs. Classical PCR confirmed amplification of both targets from cloned control templates, producing amplicons of the expected size. In probe-based real-time PCR, consistent amplification was obtained for the ASFV-3 system using a pET-asf-3 clone across serial dilutions (Ct ~27–34), while the ASFV-1 probe-based assay did not generate a detectable signal under the applied conditions. Under the tested conditions, the preliminary limit of detection observed for the ASFV-3 probe-based assay was 10³ copies per reaction. These results demonstrate the feasibility of the developed approach and indicate that the ASFV-3 system supports further analytical evaluation, while the ASFV-1 system requires additional optimization of probe parameters and/or thermal cycling conditions.