A SYBR green I-based multiplex real-time PCR for simultaneous detection of pseudorabies virus, porcine circovirus 3 and porcine parvovirus

Figures

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  Fig. 1. Melting curve analysis of the SYBR Green I-based real-time PCR assay. A PRV was detected with specific amplification of the Tm value at 90 °C. B PPV was detected with a specific amplification of the Tm value at 84 °C. C PCV3 was detected with a specific amplification of the Tm value at 80 °C

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  Fig. 2. Preparation of the plasmid standards. A-C Amplifaction curves (X-axis: cycle, Y-axis: fluorescence) of PRV, PPV and PCV3 for each plasimid standard with concentrations from 4.67 × 108 to 4.67 × 102 copies/μL, 3.67 × 108 to 3.67 × 102 copies/μL and 3.07 × 109 to 3.07 × 102 copies/μL, respectively. D-F Standard curves of PRV, PPV and PCV3 plasmid standards

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  Fig. 3. Melting curve analysis of the specificity of multiplex real-time PCR assay. Viral nucleic acids including PRV, PPV, CSFV, PRRSV, PCV3 and PCV2, were tested by multiplex real-time PCR. From the fluorescence signals collected, PRV, PPV and PCV3 had specific melting peaks. However, other viruses showed nonspecific melting peaks (ns.), but these can be separated from the specific melting curve peaks

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  Fig. 4. Sensitivity of the multiplex real-time PCR and conventional PCR assay. pMD-PRV (A, D), pMD-PPV (B, E), pMD-PCV3 (C, F), and a mixture of three standard plasmids (G, H) were used as templates and diluted from 4.67 × 106 to 4.67 × 100 copies/μL, 3.67 × 106 to 3.67 × 100 copies/μL, 3.07 × 106 to 3.07 × 100 copies/μL, and 1.87 × 107 to 1.87 × 101 copies/μL, respectively. A-C were the detection limits of PRV, PPV or PCV3 in multiplex real-time PCR reaction system. D-F were the detection limit of the conventional PCR. G-H were the detection limits of a mixture of three standard plasmids in multiplex real-time PCR reaction system. The gels were cropped. Full-length gels are presented in Fig. S1. M, DL500 marker; NC, negative control