Establishment of a Duplex Quantitative PCR Assay for the Detection and Differentiation of African Swine Fever Virus Genotype I, Genotype II, and Genotype I/II Recombinants

Figures

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  Figure 1. Schematic representation of the CP2475L in the recombinant ASFV strain VNUA/rASFV/PT2/2023 (GenBank accession no. PP810980) and positions of the duplex qPCR primer–probe sets. The CP2475L region in this strain comprises two genotype II–derived flanking regions (112,097–114,683 bp and 117,648–119,524 bp) and a central genotype I–derived region (114,684–117,647 bp), shown as boxes. Panel (A) shows the genotype II/recombinant–specific primer–probe set, along with alignments of the corresponding genome sequences from registered genotype I (10 strains), II (8 strains), genotype I/II recombinant viruses (11 strains) and one genotype X strain. Panel (B) similarly shows the genotype I/recombinant–specific primer–probe set. The primer and probe binding regions are shaded to highlight their positions in the genotype I, genotype II, and recombinant ASFV sequences. Red letters indicate mismatched nucleotides compared with the corresponding primer or probe sequences. Arrowheads indicate putative recombination breakpoints.

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  Figure 2. Geographic distribution of clinical sample collection sites in northern Vietnam. Pink-shaded areas indicate the provinces where samples were collected.

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  Figure 3. Standard amplification curves of the duplex qPCR assay generated using 10-fold serial dilutions of a positive control plasmid mixture containing equal copy numbers (100 to 106 copies per reaction) of pGEM-TV-GI and pGEM-TV-GII. Panel (A) shows the standard curve obtained using the genotype I–specific primer–probe set, and Panel (B) shows the standard curve using the genotype II-specific primer–probe set.

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  Figure 4. Specificity of the duplex qPCR assay. (A) E75 (genotype I), (B) Armenia07 (genotype II), (C) recombinant-mimicking sample (a mixture of E75 and Armenia07), and (D) Kenya05/Tk-1 (genotype X). E75 and Armenia07 each showed single-target amplification, whereas the mixed sample exhibited dual-target amplification. No amplification was observed for Kenya05/Tk-1.

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  Figure 5. Phylogenetic analysis of ASFV whole-genome sequences. A maximum-likelihood phylogenetic tree was constructed from whole-genome sequences of ASFV using the GTR + I model with 1000 bootstrap replicates. The 10 recombinant ASFVs analyzed in this study (red dots) clustered within the genotype I/II recombinant group. Bootstrap values are shown at major nodes. The scale bar indicates the number of substitutions per site.