Specific Detection of African Swine Fever Virus Variants: Novel Quadplex Real-Time PCR Assay with Internal Control

African swine fever (ASF), a highly contagious and lethal viral disease, continues to devastate the global swine industry. The emergence of ASF virus (ASFV) variants with varying genomic deletions poses significant challenges for ASF control. This study presents a novel, sensitive, and reliable quadplex real-time PCR assay for detecting ASFV variants lacking key genes (<i>I177L</i>, <i>EP402R</i>, and <i>MGF360-14L</i>), either individually or in combination. The assay targets conserved regions within these genes, ensuring broad coverage of diverse ASFV genotypes. A porcine <i>beta-actin</i> (<i>ACTB</i>) internal control was incorporated to minimize false-negative results. Optimization and evaluation using spike-in tests demonstrated high sensitivity, with a limit of detection (LOD) ranging from 1 to 10 plasmid copies or 0.1 TCID₅₀ of ASFV isolates per reaction. No cross-reactivity was observed when testing serum samples from pigs infected with other common swine viruses. Further validation across a diverse panel of samples, including those from naturally ASFV-infected field pigs (<i>n</i> = 54), experimentally ASFV-infected pigs (<i>n</i> = 50), PBS-inoculated pigs (<i>n</i> = 50), ASFV-free field pigs (<i>n</i> = 100), and feral pigs (<i>n</i> = 6), confirmed 100% specificity. This robust assay provides a valuable tool for rapid and accurate ASF surveillance and control efforts, facilitating the timely detection and mitigation of outbreaks caused by emerging ASFV variants.