Porcine alveolar macrophages (PAMs) were prepared from bronchoalveolar lavage and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, United States) supplemented with 10% fetal bovine serum (FBS, Sigma, United States), 100 U/ml penicillin, 100 μg/ml streptomycin and 250 ng/ml amphotericin B (Beyotime Biotechnology, China) at 37°C with 5% CO₂. ASFV (CSTR: 16533.06. IVCAS 6.7494, genotype II) was stored at −80°C in the biosafety level 3 (BSL-3) facility of Wuhan Institute of Virology, Chinese Academy of Sciences (WIV-CAS). All the experiments involving infectious ASFV were performed in the BSL-3 laboratory. The titer of ASFV stocks were determined by the hemadsorbing (HAD) test. Briefly, 4 × 10⁴ cells/well of PAMs were seeded into 96-well plates and infected with 10-fold diluted ASFVs. After 1-day infection, 1% porcine erythrocyte cell suspensions stored in PBS (Gibco, United States) were added into each well. The phenomena of hemadsorption were observed over 7 days by a microscope. The 50% hemadsorbing dose (HAD₅₀) was calculated by the Reed and Muench method (Zhao et al., 2019).

Reagents used to develop the assays and perform viability experiments were purchased from different companies. In summary, the PMAxx (40,069, 20 mM in H₂O) was purchased from Biotium (United States), TritonX-100 from Sigma-Aldrich (United States), Virkon™ S from DuPont (United States), and Disinfectant Basi containing 4.0–4.99% (w/v) chlorine from Yiheng (Dezhou, China). Primers and probes were synthesized by Sangon Biotech (Shanghai, China). All other chemical reagents used in the experiments were purchased from Sinopharm (Shanghai, China) except otherwise stated. Double distilled water was used in all experiments.

Extracted ASFV DNA using the Blood viral DNA extraction kit (Qiagen, catalog 51104) and virus suspensions before and after inactivation at different conditions were used to optimize the conditions of the PMAxx-qPCR assay. As shown in Figure 1, PMAxx (0, 5, 10, 25, 50, and 100 μM) together with Triton X-100 (5, 1, 0.1, 0.01%, and 0) were added into the samples. The mixtures were then incubated in the dark at room temperature (22–26°C) for 10 min. Subsequently, the mixtures were exposed to photolysis at different times (5, 10, 15, or 20 min) using a PMA-Lite^™ LED photolysis device (Biotium, United States). The photolyzed samples were heat treated at 95°C for 5 min prior to DNA extraction. Extracted DNA samples were finally detected by qPCR to determine the cycle threshold (Ct) values of the mixtures. Samples without PMAxx treatment served as positive controls. Each condition was performed in triplicate.

Nucleic acids of the samples were extracted using the Blood viral DNA extraction kit. The primer pairs and probes targeting the ASFV-P72 gene are listed in Table 2. The qPCR reaction system (total 20 μl) consisted of 5 μl template DNA, 10 μl 2 × reaction mix (Luna^® Universal Probe qPCR Master Mix, M3004S, NEB, United States), 0.4 μM forward primer, 0.4 μM reverse primer, 0.2 μM probe, and DNase free water. The qPCR reaction was performed on a Biorad CFX96 Real-Time PCR System (Bio-Rad, United States) with a denaturation step at 95°C for 1 min, followed by 45 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 30 s.

The ΔCt value was used to estimate the risk and presence of infectious ASFV in tested samples. To obtain the ΔCt value, the average Ct value of a sample after PMAxx pretreatment was subtracted from the average Ct value of the same sample without PMAxx pretreatment. Resultant data was graphically presented and statistically analyzed by GraphPad Prism version 8 (GraphPad software, United States) software. A t-test was used to test the impact of variables and determine the significant differences. Ordinary one-way ANOVA test was used to analyze the significant differences of the data among different groups. A P-Value of p < 0.05 was deemed significant.

Three main factors that may affect the ΔCt of the PMAxx-qPCR assay include the PCR amplicon size, PMAxx concentration, and photolysis time. As shown in Figure 2A, ΔCt values of the free ASFV DNA amplified using the primer/probe set #3 (amplicon size 189) were higher than those of primer/probe sets #1 (amplicon size 75) or #2 (amplicon size 163), indicating that longer amplicons were better for discrimination. Therefore, the primer/probe set #3 was used in further optimization experiments. Further tests on two types of samples (free DNA and PBS-diluted ASFV positive swine plasma) showed that PMAxx concentrations ranging from 5 μM to 100 μM (Figure 2B) and the photolysis time ranging from 5 min to 20 min (Figure 2C) had no significant difference in the ΔCt values. However, considering the fact that large amounts of nucleic acids of other organisms may be present in real life samples, a relatively high PMAxx concentration of 25 μM and longer photolysis time of 15 min was chosen for the following experiments. These conditions were also found not to have any significant interference with infectious virions when determined by cell culture as seen in Supplementary Table S1.

Heat treatment is an important method of inactivating ASFV, and it has been reported that ASFV can be inactivated after heating at temperatures higher than 60°C for 20 min (Mazur-Panasiuk et al., 2019). Using this analogy, infectious ASFV samples were heat inactivated at temperatures ≥60°C, subjected to PMAxx, and results compared to their respective control samples (without heat treatment). Compared to the control, the ΔCt values were 1.6, 2.19, 3 and 11.43 for the infectious sample and samples subjected to 60°C, 70°C and 95°C temperatures, respectively (Figure 3A). These results indicated that the PMAxx-qPCR assay could optimally determine inactivated viruses heated at higher temperatures of ≥95°C, but not at mild temperatures (60°C or 70°C). The probable reason for this dismal performance at mild temperatures was thought to be related to the existence of intact ASFV capsid structures not easily broken by mild temperatures.

It has been reported that surfactants such as Triton X-100 (Coudray-Meunier et al., 2013) and SDS (Hong et al., 2021) can increase the permeability of monoazide dyes to pathogenic viruses with intact viral capsids. Therefore, we attempted to add Triton X-100 to enhance the penetration of PMAxx into the intact ASFV virions inactivated at 60°C. As shown in Figure 3B (left), Triton X-100 (5, 1, 0.1, 0.01% (v/v)) did not promote PMAxx penetration into the control (infectious ASFV viruses without heat treatment), but it increased the PMAxx penetration into ASFV samples inactivated at 60°C (Figure 3B, right). Further analysis of the ΔCt values of inactivated samples showed that 0.1% Triton X-100 had the largest ΔCt value (Figure 3B, right) and was hence chosen as an assistant to the PMAxx-qPCR assay in the following tests.

Using this concentration, further testing on serial dilutions of infectious ASFV suspensions (Figure 3C) showed that there was no significant difference between the Triton X-100-qPCR alone (without PMAxx treatment) and the Triton X-100 assisted PMAxx-qPCR. These results indicated that the Triton X-100 assisted PMAxx treatment had no effects on infectious ASFV. However, after heat treatment at 60°C for 20 min (Figure 3D), the Ct values of the qPCR did not change, but the Ct values of the Triton X-100 assisted PMAxx-qPCR increased significantly (p < 0.001 for all groups), showing no amplification after 40 cycles except for the highest concentration of 5.7log₁₀HAD₅₀/mL (Ct value: 37.96 ± 1.83).

In order to verify if the Triton X-100 assisted PMAxx-qPCR assay could discriminate infectious ASFV in partially inactivated samples, a series of samples were prepared by mixing the infectious 5.32log₁₀HAD₅₀/mL ASFV and heat-inactivated 5.7 log₁₀HAD₅₀/mL ASFV at different ratios. As shown in Table 3, an increase in the percentage of infectious ASFV in the samples led to a decrease in ΔCt values. Even at 1% infectious ASFV in the samples, the ΔCt values were significantly lower than those of the ΔCt values of 100% inactivated ASFV. The same trends were observed even with lower titers of ASFV mixtures. These results demonstrated the possibility of using the Triton X-100 assisted PMAxx-qPCR assay to determine small percentages of infectious ASFV in samples by comparing the difference between the ΔCt value of the sample before inactivation and that of the same sample inactivated at 60°C for 20 min.

The detection of ASFV varies in different bio-matrices, this may possibly affect the performance of Triton X-100 assisted PMAxx-qPCR assays. To determine this, samples with inactivated ASFV suspended in five bio-matrices [PBS, swine blood, swine tissue homogenate, pig saliva swab (SS), and environmental swabs (ES)] were tested using both qPCR (without PMAxx treatment) and Triton X-100 assisted PMAxx-qPCR. Compared to PBS at 1× concentration, late Ct values were observed in blood and saliva matrices when detected by qPCR (Table 4). However, after adding the inactivated ASFV into 4 × or 8 × PBS-diluted matrices, early Ct values were observed. These results indicated that these two matrices would affect either the efficacy of the DNA extraction kits or contain some inhibitors that might inhibit the qPCR reaction. Additionally, blood may also affect the PMAxx treatment process since a late Ct value was observed in the undiluted blood when detected by the Triton X-100 assisted PMAxx-qPCR assay. Therefore, blood and saliva samples need to be diluted with PBS at least 4× or 8× times before detection, while tissue homogenates and the environmental swabs do not need any dilutions.

In order to evaluate whether the Triton X-100 assisted PMAxx-qPCR assay is suitable for detecting viable ASFV after chemical disinfection, ASFV inactivated by five types of chemical disinfectants (NaClO, GA, HAc, NaOH, and VK) at different concentrations was tested, with ddH₂O treatment serving as a positive control. Cell culture was also used to determine if there remained infectious ASFV after the disinfections. As shown in Figure 4A, the cell culture revealed that only the H₂O-treatment group contained the infectious ASFV and no growth of ASFV could be detected after the chemical disinfections. The Triton X-100 assisted PMAxx-qPCR assay also revealed that there existed infectious ASFV in the H₂O-treatment group because the ΔCt value was only 0.37 (p < 0.05). However, for the chemical disinfection groups, the Ct values of qPCR (without PMAxx treatment) alone were significantly increased except for the NaOH-treated group (Figure 4B). The increase in Ct value may be a result of the four chemical disinfectants degrading or covalently cross-linking with ASFV DNA, especially GA. After the Triton X-100 assisted PMAxx treatment, the Ct values of these chemical groups increased further to above 37 or no amplification after 40 cycles (Figure 4B). This signified that the chemicals were indeed active against ASFV.