Real-time quantitative PCR (qPCR) has been widely used for African swine fever virus (ASFV) surveillance, but it cannot differentiate viable from inactivated virions, as inactivated particles often retain intact nucleic acids. This limitation may lead to overestimation of the presence of viruses in tested samples, potentially resulting in unnecessary outbreak responses and increased economic burdens. To address this challenge, a viability qPCR (V-qPCR) assay was established, utilizing nucleic acid intercalating dyes-propidium monoazide (PMA) or ethidium monoazide (EMA), to discriminate viable and inactivated ASFV. These dyes selectively penetrate compromised viral envelopes in inactivated particles, covalently binding viral DNA, and inhibiting PCR amplification. The optimized V-qPCR assay demonstrated a detection limit of 10^1.5 TCID₅₀/mL for infectious ASFV and effectively discriminated chemically inactivated virus (10^4.5 TCID₅₀/mL) treated with potassium peroxymonosulfate (PPMS), sodium hydroxide (NaOH), or acetic acid (HAc), showing no amplification signals. In simulated environmental samples (ASFV-spiked swabs from farm walls, rails, feed, floors, urine, and feces), the V-qPCR assay reliably detected inactivation in PPMS-treated samples (walls, rails, urine, feces) and NAOH-treated samples (walls, feces), showing no amplification. In contrast, HAc-inactivated samples yielded residual amplification (Ct ~ 31.5), suggesting superior performance of PPMS and NAOH in complex matrices. Validation using 30 ASFV-positive field environmental swabs revealed three outcomes: (i) 21 samples showed no amplification (confirmed inactivated); (ii) six samples showed similar Ct values before and after heat treatment (ΔCt [Ct(_+dye)-Ct(_{no dye})] < 2.5, indicating inactivation); and (iii) three samples demonstrated detectable Ct values before heat inactivation but no amplification after treatment (confirming the presence of viable virus). This study represents the first application of V-qPCR for field-based assessment of ASFV infectivity, offering a rapid and practical tool to enhance diagnostic accuracy and biosecurity management in pig farms.

Keywords: African swine fever virus; EMA; PMA; pathogen inactivation; viability quantitative PCR; viral presence.