Development of a triplex real-time quantitative PCR for detection and differentiation of genotypes I and II African swine fever virus

Figures

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  Figure 1. The locations of the specific primers and probes designed for the triplex qPCR. The nucleotide sequence alignments of the partial B646L gene (A), F1055L gene (B), and E183L gene (C) of ASFV show the locations of the primers and probes. F, P, and R indicate the forward primer, TaqMan probe, and reverse primer, respectively. “·” indicates the same nucleotide acid and “-” indicates the deletion of nucleotide acid.

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  Figure 2. The amplification curves of the recombinant plasmid constructs generated for 24 genotypes of ASFV. The recombinant plasmid constructs were used as templates to validate the specific primers and probe ASFV-B646L-F/R/P. 1: pASFV-I; 2: pASFV-II; 3: pASFV-III; 4: pASFV-IV; 5: pASFV-V; 6: pASFV-VI; 7: pASFV-VII; 8: pASFV-VIII; 9: pASFV-IX; 10: pASFV-X; 11: pASFV-XI; 12: pASFV-XII; 13: pASFV-XIII; 14: pASFV-XIV; 15: pASFV-XV; 16: pASFV-XVI; 17: pASFV-XVII; 18: pASFV- XVIII; 19: pASFV-XIX; 20: pASFV-XX; 21: pASFV-XXI; 22: pASFV-XXII; 23: pASFV-XXIII; 24: pASFV-XXIV; 25: negative clinical sample control; 26: RNase-free distilled water control.

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  Figure 3. Generation of the standard curves. The amplification curves of pASFV-B646L (A), pASFV-E183L (B), pASFV-F1055L (C), and their standard curves (D) are shown. 1–7: the final concentrations of the standard plasmid constructs ranged from 1.55 × 107 to 1.55 × 101 copies/μL.

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  Figure 4. Specificity analysis. 1a: ASFV genotype II B646L gene; 1b: ASFV genotype II E183L gene; 2a: ASFV genotype I B646L gene; 2b: ASFV genotype I F1055L gene; 3–9: PRRSV, PEDV, FMDV, CSFV, SIV, PRV, PCV2, respectively; 10: negative clinical sample control; 11: RNase-free distilled water control. In the figure, the red amplification curves correspond to primers and probe targeting ASFV B646L gene, the blue amplification curve corresponds to the primers and probe targeting ASFV E183L gene, and the green amplification curve corresponds to the primers and probe targeting ASFV F1055L gene.

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  Figure 5. Sensitivity analysis. 1–9: the final concentrations of the standard plasmid constructs pASFV-B646L (A), pASFV-E183L (B), and pASFV-F1055L (C) ranged from 1.55 × 107 to 1.55 × 10−1 copies/μL. 10: negative control (RNase-free distilled water).

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  Figure 6. PROBIT regression results to determine the analytical sensitivity. Ct values and hit rates were measured using ten-fold serial dilutions of standard plasmid constructs. The LODs of pASFV-B646L (A), pASFV-F1055L (B), and pASFV-E183L (C) were determined to be 399.647 copies/reaction (95% confidence interval 339.947–554.984 copies/reaction), 374.409 copies/reaction (95% confidence interval 316.95–492.742 copies/reaction), and 355.083 copies/reaction (95% confidence interval 294.493–468.449 copies/reaction), respectively.

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  Figure 7. The representative amplification curves of ASFV genotype I positive sample (A), genotype II positive sample (C), and mixture of genotypes I and II positive sample (B). 1: ASFV B646L gene; 2: ASFV F1055L gene; 3: ASFV E183Lgene; 4: negative clinical sample control; 5: RNase-free distilled water control.