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

Figures

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  Figure 1. Schematic diagram of V-qPCR for discriminating viable and inactivated ASFV. PMA or EMA (represented as red dots) selectively penetrates damaged membranes of inactivated ASFV virions. Upon photoactivation, the dyes covalently cross-link with viral DNA, thereby inhibiting qPCR amplification. In contrast, the genomic DNA from viable ASFV remains accessible and is normally amplified.

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  Figure 2. Optimization of the working conditions of the V-qPCR assay for detecting heat-inactivated ASFV. A, B Optimization of PMA and EMA dye concentrations. C, D Optimization of dark incubation time. E, F Optimization of photolysis time. *P < 0.05, **P < 0.01; ***P < 0.001; ns, not significant.

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  Figure 3. Effects of surfactants on the performance of V-qPCR in discriminating viable and inactivated ASFV. A V-qPCR detection of heat-inactivated ASFV (104.5 TCID50/mL) in the presence of different concentrations (0.0001%, 0.001%, and 0.01%) of triton X-100, tween 20, or SDS. Dye-treated PBS without surfactant served as the control. B Effects of surfactants on qPCR detection of infectious and heat-inactivated ASFV in the absence of dye, with PBS as a control. C Effects of SDS on V-qPCR detection of viable and inactivated ASFV, with dye-treated PBS without SDS as a control. Unfilled columns represent no amplification and Ct values were set to 40 for ΔCt calculation. *P < 0.05, **P < 0.01; ***P < 0.001; ns, not significant.

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  Figure 4. Specificity and sensitivity of V-qPCR for detecting ASFV. A, B Specificity testing of the V-qPCR assay against other common porcine viruses, including CSFV, PRRSV, PRV, PCV2, and APPV. C, D Sensitivity testing of the V-qPCR assay using serial dilutions of infectious ASFV (104.5 to 100.5 TCID50/mL). PBS was used as a negative control.

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  Figure 5. Evaluation of V-qPCR for detecting ASFV inactivated by thermal treatment or chemical disinfectants. A Comparison of qPCR and V-qPCR for detecting ASFV inactivated by heat treatment (60 °C, 90 °C, or 105 °C for 60 min; initial titer 104.5 TCID50/mL). B − G Optimization of PMA and EMA dye concentrations for detecting ASFV inactivated by disinfectants PPMS (B, C), NaOH (D, E), or HAc (F, G). H Comparison of qPCR and V-qPCR for detecting ASFV inactivated by PPMS, NaOH, or HAc. After inactivation, samples were tenfold diluted with PBS prior to analysis. Data are presented as mean ± SD of three independent replicates. Unfilled columns indicate no amplification and Ct values were set to 40 for ΔCt calculation. *P < 0.05, **P < 0.01; ***P < 0.001; ns, not significant.

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  Figure 6. Evaluation of the effects of environmental sample matrices on viable ASFV detection using V-qPCR. A, B Infectious ASFV was spiked into negative environmental samples (swabs from walls, rails, feed, floors, urine, and feces) collected from pig farms to a final titer of 104.5 TCID50/mL. Samples were then tenfold diluted with PBS and detected by qPCR and V-qPCR. *P < 0.05, **P < 0.01; ***P < 0.001; ns, not significant.

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  Figure 7. Performance of V-qPCR for differentiating viable and inactivated ASFV in simulated environmental samples. A − H Detection of simulated environmental samples inactivated by 105 °C (A, B), PPMS (C, D), NaOH (E, F), or HAc (G, H) using qPCR and V-qPCR. Infectious ASFV was spiked into negative environmental swabs from walls, rails, feed, floors, urine, and feces to a final titer of 104.5 TCID50/mL, and inactivated. Samples were then tenfold diluted with PBS before analysis. Unfilled columns represent no amplification and Ct values were set to 40 for ΔCt calculation. †, one of the three repeats with no amplification. ‡, two of the three repeats with no amplification. *P < 0.05, **P < 0.01; ***P < 0.001; ns, not significant.