Development and Application of a Multiplex Real-Time TaqMan qPCR Assay for the Simultaneous Detection of African Swine Fever Virus, Classical Swine Fever Virus, Porcine Reproductive and Respiratory Syndrome Virus, Pseudorabies Virus, and Porcine Circovirus Type 2

Figures

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  Figure 1. Optimization of fluorescence intensity for the multiplex qPCR assay. Effects of primer concentrations (0.12–0.40 pmol/μL) on fluorescence signals for ASFV (A), CSFV (B), PRRSV (C), PRV (D), and PCV2 (E) detection. Effects of probe concentrations (0.08–0.36 pmol/μL) on fluorescence signals for ASFV (F), CSFV (G), PRRSV (H), PRV (I), and PCV2 (J) detection. Effects of annealing temperature gradients (46–60°C) on fluorescence signals for ASFV (K), CSFV (L), PRRSV (M), PRV (N), and PCV2 (O) detection. Optimization was performed using recombinant plasmid standards as templates on an ABI 7500 Real-Time PCR System (Thermo Fisher Scientific, USA).

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  Figure 2. Detection standard curves of the multiplex real-time PCR method developed in this study. Under optimal amplification conditions, the optimized ASFV (A), CSFV (B), PRRSV (C), PRV (D), and PCV2 (E) amplification curves were generated using real-time PCR. Linear regression analysis of standard curve of ASFV (F), CSFV (G), PRRSV (H), PRV (I), and PCV2 (J) generated from 10-fold serial dilutions of plasmid standards (1 × 104–1 × 108 copies/μL).

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  Figure 3. Specificity of the multiplex real-time PCR assay. Amplification curves were generated for the following targets: 1: ASFV; 2: CSFV; 3: PRRSV; 4: PCV2; 5: PRV; 6: Porcine epidemic diarrhea virus (PEDV); 7: TGEV; 8: PPV; 9: Nuclease-free H2O. No cross-detection was observed, as only target-specific plasmids generated amplification signals in their respective fluorescence channels (FAM/ASFV, NED/CSFV, VIC/PRRSV, ROX/PCV2, CY5/PRV), confirming primer-probe specificity. Tests were performed using plasmid standards for target viruses and extracted nucleic acids from non-target pathogens under optimized assay conditions (56 °C annealing temperature, 0.32 pmol/μL primers, 0.12 pmol/μL probes).

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  Figure 4. Sensitivity of the multiplex real-time PCR assay. Detection limit for (A) P72 gene of ASFV; (B) 5′UTR of CSFV; (C) OFR6 gene of PRRSV; (D) gB gene of PRV; (E) cap gene of PCV2. Serial 10-fold dilutions of mixed plasmid standards (1 × 100–1 × 108 copies/μL) were tested. The limit of detection was 10 copies/μL for all targets.