Establishment of a Triplex qPCR Assay for Differentiating Highly Virulent Genotype I Recombinant Virus From Low-Virulence Genotype I and Genotype II African Swine Fever Viruses Circulating in China

Abstract

African swine fever virus (ASFV) poses serious threats to the global swine industry, food safety, and the economy. Since August 2018, different types of ASFVs have successively emerged in China, making ASF diagnostics more challenging. The highly virulent genotype I recombinant virus has gradually become the prevalent dominant strain and is identified by sequencing several of its genes, which is time-consuming and expensive. Here, we developed a triplex real-time quantitative PCR (qPCR) assay based on the ASFV B646L, X64R, and MGF_360-14L genes to differentiate highly virulent genotype I recombinant viruses from low-virulence genotype I and genotype II viruses in China. This method has high sensitivity and a limit of detection of 10 copies/reaction for standard plasmids, as well as good specificity without cross-reactions with the viral nucleic acids of porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine circovirus 2 (PCV 2), porcine circovirus 3 (PCV 3), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), or porcine rotavirus (PoRV). Importantly, triplex qPCR can be used to quickly and accurately evaluate clinical samples and cell cultures infected with highly virulent genotype I virus, low-virulence genotype I virus, or genotype II virus. Thus, triplex qPCR provides an alternative tool for ASF surveillance in China.

Keywords: African swine fever virus; differential detection; genotype II; highly virulent genotype I; low virulent genotype I; triplex real-time quantitative PCR.

Figure 1

Sequences and locations of the primers and probes used for triplex qPCR. The nucleotide sequences of nine highly virulent genotype I ASFVs, five low-virulence genotype I ASFVs, and six genotype II ASFVs are presented to show the primers and probes specific for the B646L gene (A), the X64R gene (B), and the MGF_360-14 L gene (C). The positions of the primers and probes are indicated by the locations in the genome of ASFV JS/LG/21. Dots (.) indicate identical bases. Gaps represent gene deletions in the viral genome. F and R indicate the forward primer and reverse primer, respectively. MGF, the MGF_360-14 L gene. S, C/G bases and Y, C/T bases in the MGF-F primer.

Figure 2

Establishment of triplex qPCR. The fluorescence signals of (A) FAM, VIC, and CY5 for the highly virulent genotype I virus JS/LG/21; (B) of FAM and VIC for the low virulent genotype I virus SD/DY-I/21; and of FAM and CY5 for (C) the highly virulent genotype II virus HLJ/18 and (D) the lower virulent genotype II virus HLJ/HRB1/20 were monitored via triplex qPCR. No fluorescent signal was detected from the negative control (ddH₂O, not shown in the diagram). The RFUs of different cycles are expressed as the means of three replicates from a sample in an experiment.

Figure 3

Standard curves and sensitivity of triplex qPCR. Tenfold serial dilutions ranging from 10⁶ to 10¹ copies/5 μl of the standard plasmid mixture (pCAGGS-B646L, pCAGGS-X64R, and pCAGGS-MGF) were detected via triplex PCR. Each sample was tested in triplicate. (A) Standard curves and (B) sensitivity of the assay for the B646L gene. (C) Sensitivity of the assay for the X64R gene. (D) Sensitivity of the assay for the MGF_360-14 L (MGF) gene.

Figure 4

Specificity of triplex qPCR. The FAM, VIC, and CY5 fluorescence signals were detected via triplex qPCR; (A) viral DNA of the highly virulent genotype I virus JS/LG/21, (B) the low virulent genotype I virus SD/DY-I/21, and (C) the genotype II virus HLJ/18 served as positive controls. (D) No fluorescence signal was detected when the viral genomes of PRV, PRRSV, CSFV, PEDV, PCV 2, PCV 3, TGEV, and PoRV or the negative control (ddH₂O) were used as templates. The RFUs of different cycles are expressed as the means of three replicates from a sample in an experiment.