Triplex Crystal Digital PCR for the Detection and Differentiation of the Wild-Type Strain and the MGF505-2R and I177L Gene-Deleted Strain of African Swine Fever Virus

Abstract

African swine fever (ASF) is a severe and highly contagious viral disease that affects domestic pigs and wild boars, characterized by a high fever and internal bleeding. The disease is caused by African swine fever virus (ASFV), which is prevalent worldwide and has led to significant economic losses in the global pig industry. In this study, three pairs of specific primers and TaqMan probes were designed for the ASFV B646L, MGF505-2R and I177L genes. After optimizing the reaction conditions of the annealing temperature, primer concentration and probe concentration, triplex crystal digital PCR (cdPCR) and triplex real-time quantitative PCR (qPCR) were developed for the detection and differentiation of the wild-type ASFV strain and the MGF505-2R and/or I177L gene-deleted ASFV strains. The results indicate that both triplex cdPCR and triplex qPCR were highly specific, sensitive and repeatable. The assays could detect only the B646L, MGF505-2R and I177L genes, without cross-reaction with other swine viruses (i.e., PRRSV, CSFV, PCV2, PCV3, PEDV, PDCoV and PRV). The limit of detection (LOD) of triplex cdPCR was 12 copies/reaction, and the LOD of triplex qPCR was 500 copies/reaction. The intra-assay and inter-assay coefficients of variation (CVs) for repeatability and reproducibility were less than 2.7% for triplex cdPCR and less than 1.8% for triplex qPCR. A total of 1510 clinical tissue samples were tested with both methods, and the positivity rates of ASFV were 14.17% (214/1510) with triplex cdPCR and 12.98% (196/1510) with triplex qPCR, with a coincidence rate of 98.81% between the two methods. The positivity rate for the MGF505-2R gene-deleted ASFV strains was 0.33% (5/1510), and no I177L gene-deleted ASFV strain was found. The results indicate that triplex cdPCR and triplex qPCR developed in this study can provide rapid, sensitive and accurate methods for the detection and differentiation of the ASFV B646L, MGF505-2R and I177L genes.

Keywords: African swine fever virus (ASFV); gene-deleted strain; multiplex crystal digital PCR (multiplex cdPCR); multiplex real-time quantitative PCR (multiplex qPCR); wild-type strain.

Figure 1

Optimization of the primer and probe concentrations (A–C) and the annealing temperature (D) for triplex cdPCR. (A–C) Amplification results of pASFV-B646L-1, pASFV-MGF505-2R-1 and pASFV-I177L-1 plasmids (all at final reaction concentrations of 2.0 × 10⁴ copies/µL) with different probe and primer concentrations. NC: Negative control. (D) Amplification results of pASFV-B646L-1, pASFV-MGF505-2R-1 and pASFV-I177L-1 plasmids (all at final reaction concentrations of 2.0 × 10⁴ copies/µL) with different annealing temperatures.

Figure 2

Generation of the standard curves of triplex qPCR. Amplification curves (A–C) and standard curves (D) of triplex qPCR. (A–C) Final reaction concentrations of curves 1 to 6 range from 2.0 × 10⁶ to 2.0 × 10¹ copies/μL; 8: Negative control.

Figure 3

Generation of the standard curves of triplex cdPCR. (A–C) Final reaction concentrations of the plasmids range from 2.0 × 10⁴ to 2.0 × 10⁰ copies/μL. (D) shows the standard curves.

Figure 4

Sensitivity analysis of triplex qPCR. (A–C) Final reaction concentrations of curves 1 to 7 range from 2.0 × 10⁶ to 2.0 × 10⁰ copies/μL; 8: Negative control.

Figure 5

Sensitivity analysis of triplex cdPCR. (A–C) Final reaction concentrations of the plasmids range from 2.0 × 10⁵ to 2.0 × 10⁻¹ copies/μL. NC: Negative control.

Figure 6

Specificity analysis of triplex cdPCR (A–C) and triplex qPCR (D). (A–C) Results of the specificity tests of the B646L gene (A), the MGF505-2R gene (B) and the I177L gene (C). NC: Negative control. (D) Results of the specificity test of triplex qPCR. 1a: pASFV-B646L-1; 1b: pASFV-I177L-1; 1c: pASFV-MGF505-2R-1; 2a: ASFV B646L gene; 2b: ASFV I177L gene; 2c: ASFV MGF505-2R gene; 3: pASFV-ΔI177L; 4: pASFV-ΔMGF505-2R; 5: CSFV; 6: PRRSV; 7: PCV2; 8: PCV3; 9: PEDV; 10: PDCoV; 11: PRV; 12: Negative control.