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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 (I177L, EP402R, and MGF360-14L), either individually or in combination. The assay targets conserved regions within these genes, ensuring broad coverage of diverse ASFV genotypes. A porcine beta-actin (ACTB) 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 (n = 54), experimentally ASFV-infected pigs (n = 50), PBS-inoculated pigs (n = 50), ASFV-free field pigs (n = 100), and feral pigs (n = 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.<\/jats:p>","DOI":"10.3390\/microorganisms13030615","type":"journal-article","created":{"date-parts":[[2025,3,7]],"date-time":"2025-03-07T07:05:46Z","timestamp":1741331146000},"page":"615","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Specific Detection of African Swine Fever Virus Variants: Novel Quadplex Real-Time PCR Assay with Internal Control"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7951-4175","authenticated-orcid":false,"given":"Lihua","family":"Wang","sequence":"first","affiliation":[{"name":"Center on Biologics Development and Evaluation, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA"},{"name":"Department of Anatomy and 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