Development and evaluation of duplex TaqMan real-time PCR assay for detection and differentiation of wide-type and MGF505-2R gene-deleted African swine fever viruses

Abstract

Background: African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used.

Results: In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent.

Conclusion: We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

Keywords: African swine fever virus; Differential diagnosis; Duplex real-time PCR; Gene-deleted strains.

Fig. 1

Sequence analysis and alignment of primers and probes in ASFVs. The nucleotides sequence of primers and probes are consistent among the genotype II reference ASFV strains. Compared with the genotype I reference strains, the forward primer and probe targeting B646L gene have one nucleotide mutation at A345G and T376G sites, respectively. The probe targeting MGF505-2R gene has one nucleotide mutation at T265C site

Fig. 2

Specificity of the duplex TaqMan real-time PCR assay. Other porcine viruses and the standard recombinant plasmid (pUC57-B646L and pUC57-MGF505-2R) were used to test the specificity

Fig. 3

Sensitivity of the duplex real-time PCR assay. Sensitivity of the duplex real-time PCR for B646L gene and MGF505-2R gene. a 1–9: 5.8 × 10⁸–10⁰ dilutions of pUC57-B646L plasmid. b 1–9: 3.0 × 10⁸–10⁰ dilutions of pUC57-MGF505-2R plasmid

Fig. 4

Standard curves for the duplex TaqMan PCR assay. a Standard curve for B646L gene, y = − 3.149x + 36.146, R² = 0.990. b Standard curve for MGF505-2R gene, y = − 3.223x + 37.042, R² = 0.999