Introduction: The detection of African swine fever virus (ASFV) is commonly performed using quantitative real-time PCR (qPCR), a widely used virological method known for its high sensitivity and specificity. However, qPCR has a limitation in distinguishing between infectious and inactivated virus, which can lead to an overestimation of viral targets.
Methods: To provide insights into ASFV infectivity, we evaluated the suitability of PMAxx, an improved version of propidium monoazide (PMA), as a means to differentiate between infectious and non-infectious ASFV. Pre-treatment with 50 μM PMAxx for 15 min significantly reduced the qPCR signal of ASFV in the live vaccine. Additionally, thermal treatment at 85°C for 5 min effectively inactivated the live ASFV in the vaccine. Based on a standard curve, the sensitivity of the PMAxx-qPCR assay was estimated to be approximately 10 copies/μL. Furthermore, we observed a strong agreement between the results obtained from PMAxx-qPCR and pig challenge experiments. Moreover, we utilized the PMAxx-qPCR assay to investigate the persistence of ASFV, revealing a close relationship between viral persistence and factors such as temperature and type of piggery materials.
Conclusion: The findings of this study suggest that pre-treating viruses with PMAxx prior to qPCR is a reliable method for distinguishing between infectious and non-infectious ASFV. Thus, integrating of PMAxx-qPCR into routine diagnostic protocols holds potential for improving the interpretation of positive ASFV results obtained through qPCR.
Keywords: African swine fever virus; persistence; propidium monoazide; quantitative PCR; viability.
Copyright © 2024 Li, Wang, Qing, Hu, Vo, Thi, Wang and Li.