Rapid differentiation of viable and inactivated African swine fever virus by a viability quantitative PCR

作者：G. W. Fan, Xiaowei Gao, Hang Xu, Xinyong Dong, Hao Song, Yan-Hui Fu, Jing Li, Yuying Yang, Hua‐Ji Qiu, Yuzi Luo · 发表于：Veterinary Research · 年份：2025 · DOI：10.1186/s13567-025-01641-6 · 被引用次数：2 · 研究领域：Animal Disease Management and Epidemiology、Animal Virus Infections Studies、Viral Infections and Immunology Research

Abstract 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 TCID 50 /mL for infectious ASFV and effectively discriminated chemically inactivated virus (10 4.5 TCID 50 /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...