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

Abstract

IntroductionThe 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.MethodsTo 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.ConclusionThe 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.

Rapid Determination of SARS-CoV-2 Integrity and Infectivity by Using Propidium Monoazide Coupled with Digital Droplet PCR

“…To address these points, a sample pre-treatment with DNA intercalants such as PMA and its derivatives (i.e., PMAxx) has been proposed to distinguish between infectious and non-infectious viruses. Recent studies have demonstrated the selective detection of various viruses using PMA, including norovirus, enterovirus, African swine fever viruses, and Red Sea Bream Iridovirus [ 20 , 21 , 22 , 23 ]. Recently, Hong et al [ 18 ] conducted in vitro experiments by inactivating or not inactivating the SARS-CoV-2 with UV light and using sodium dodecyl sulfate as a mild membrane destabilizer to increase the PMA permeability [ 18 ].…”

“…Therefore, only genomes from intact virions will be amplified and detected, while free nucleic acids or those from non-infectious viruses are not considered by a PCR. Initially, this test was primarily used to assess the integrity of bacterial pathogens [ 19 ]; however, it has since been applied to numerous viruses [ 16 , 17 , 18 , 20 , 21 , 22 , 23 ]. With the emergence of SARS-CoV-2, the question has arisen as to whether the use of PMA can aid in identifying viruses capable of infecting and producing new viral progeny from viral remnants or incomplete viruses and free viral genomes, particularly in samples that test positive by molecular assays but have low viral loads and stay positive for prolonged periods.…”