Application of propidium monoazide quantitative PCR to discriminate of infectious African swine fever viruses

Yang Li; Zewei Wang; Jie Qing; Dajun Hu; Hong Trang Vo; Kim Thanh Thi; Xinglong Wang; Xiaowen Li

doi:10.3389/fmicb.2023.1290302

Application of propidium monoazide quantitative PCR to discriminate of infectious African swine fever viruses

Front Microbiol. 2024 Jan 10:14:1290302. doi: 10.3389/fmicb.2023.1290302. eCollection 2023.

Authors

Yang Li^#¹, Zewei Wang^#¹, Jie Qing¹, Dajun Hu², Hong Trang Vo², Kim Thanh Thi², Xinglong Wang³, Xiaowen Li^{1

2

3}

Affiliations

¹ Xiajin New Hope Liuhe Agriculture and Animal Husbandry Co., Ltd., (Shandong Engineering Laboratory of Pig and Poultry Healthy Breeding and Disease Diagnosis Technology), Dezhou, China.
² New Hope Binh Phuoc livestock Co., Ltd., Huyen Hon Quan, Vietnam.
³ College of Veterinary Medicine, Northwest A&F University, Xianyang, Yangling, China.

^# Contributed equally.

Abstract

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.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Taishan Industry Leadership Talent Project of Shandong Province in China.