The Development of a Multiplex Real-Time Quantitative PCR Assay for the Differential Detection of the Wild-Type Strain and the MGF505-2R, EP402R and I177L Gene-Deleted Strain of the African Swine Fever Virus

Abstract

African swine fever virus (ASFV) causes African swine fever (ASF), a devastating hemorrhagic disease of domestic pigs and wild boars. Currently, the MGF505R, EP402R (CD2v) and I177L gene-deleted ASFV strains were confirmed to be the ideal vaccine candidate strains. To develop an assay for differentiating the wild-type and gene-deleted ASFV strains, four pairs of specific primers and TaqMan probes targeting the ASFV B646L (p72), I177L, MGF505-2R and EP402R (CD2v) genes were designed. A multiplex real-time qPCR assay for the differential detection of the wild-type and gene-deleted ASFV strains was developed after optimizing the reaction conditions, including the annealing temperature, primer concentration and probe concentration. The results showed that the multiplex real-time qPCR assay can specifically test the ASFV B646L (p72), I177L, MGF505-2R and EP402R (CD2v) genes with a limit of detection (LOD) of 32.1 copies/μL for the B646L (p72) gene, and 3.21 copies/μL for the I177L, MGF505-2R and EP402R (CD2v) genes. However, the assay cannot test for the classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine circovirus type 2 (PCV2), PCV3 and pseudorabies virus (PRV). The assay demonstrated good repeatability and reproducibility with coefficients of variation (CV) less than 1.56% for both the intra- and inter-assay. The assay was used to test 4239 clinical samples, and the results showed that 12.60% (534/4239) samples were positive for ASFV, of which 10 samples lacked the EP402R gene, 6 samples lacked the MGF505-2R gene and 14 samples lacked the EP402R and MGF505-2R genes. The results indicated that the multiplex real-time qPCR developed in this study can provide a rapid, sensitive and specific diagnostic tool for the differential detection of the ASFV B646L, I177L, MGF505-2R and EP402R genes.

Keywords: African swine fever virus (ASFV); detection; differentiation; gene-deleted strain; multiplex real-time qPCR; wild-type strain.

Figure 1

Dynamic curves and standard curves of the multiplex real-time qPCR: The dynamic curves were generated by using the recombinant standard plasmids pASFV-B646L (A), pASFVI177L (B), pASFV-MGF505-2R (C) and pASFVEP402R (D). The standard curves (E) were generated from the dynamic curves. In (A–D), the plasmid concentrations of curves 1 to 7 ranged from 3.21 × 10⁸ copies/µL to 3.21 × 10² copies/µL (final concentration).

Figure 1

Dynamic curves and standard curves of the multiplex real-time qPCR: The dynamic curves were generated by using the recombinant standard plasmids pASFV-B646L (A), pASFVI177L (B), pASFV-MGF505-2R (C) and pASFVEP402R (D). The standard curves (E) were generated from the dynamic curves. In (A–D), the plasmid concentrations of curves 1 to 7 ranged from 3.21 × 10⁸ copies/µL to 3.21 × 10² copies/µL (final concentration).

Figure 2

Specificity analysis of the multiplex real-time qPCR: 1a: pASFV-B646L; 1b: pASFV-I177L; 1c: pASFV-MGF505-2R; 1d: pASFV-EP402R; 2: pASFV-ΔI177L; 3: pASFV-ΔMGF505-2R; 4: pASFV-ΔEP402R; 5: CSFV; 6: PRRSV; 7: PCV2; 8: PCV3; 9: PEDV; 10: PDCoV; 11: PRV; 12: Negative control.

Figure 3

Sensitivity analysis of the multiplex real-time qPCR: The dynamic curves were generated by using the recombinant standard plasmids pASFV-B646L (A), pASFVI177L (B), pASFV-MGF505-2R (C) and pASFVEP402R (D). In (A–D), the plasmid concentrations of curves 1 to 10 ranged from 3.21 × 10⁸ copies/µL to 3.21 × 10⁻¹ copies/μL (final concentration); 11: Negative control.

Figure 4

Evaluation of the clinical samples by the multiplex real-time qPCR: (A) ASFV-positive sample that lacked MGF505-2R and EP402R genes; (B) ASFV-positive sample that lacked EP402R gene; (C) ASFV-positive sample that lacked MGF505-2R gene. Curves 1a, 1b, 1c and 1d are the dynamic curves of the standard plasmids pASFV-B646L, pASFV-I177L, pASFV-MGF505-2R and pASFV-EP402R, respectively, which were used as positive controls. Curves 2a, 2b, 2c and 2d are the dynamic curves of the ASFV B646L, I177L, MGF505-2R and EP402R genes in the clinical samples, respectively. Curve 3: Negative control.