Use of dual priming oligonucleotide system-based multiplex RT-PCR assay to detect five diarrhea viruses in pig herds in South China

Figures

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  Fig. 1. Development of a single DPO RT-PCR assay for detection of SADS-CoV, TGEV, PEDV, PDCoV, and PRV-A. M, Marker; Lane 1, SADS-CoV; Lane 2, TGEV; Lane 3, PEDV; Lane 4, PDCoV; Lane 5, PRV-A

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  Fig. 2. Optimization of the DPO system-based multiplex RT-PCR primer concentration. Lanes 1–8 represent the 8 primer concentrations 0.025 μM, 0.05 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, and 0.6 μM; lane 9 represents negative control (N)

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  Fig. 3. Annealing temperature optimization comparison between DPO primers and conventional primers. Lanes 1–6 represent 40 °C, 44 °C, 48 °C, 52 °C, 56 °C, 60 °C, for a gradient with 6 temperatures; Lane 7 represents negative template (N). a DPO primer annealing temperature optimization test; b Normal annealing temperature optimization test for primers

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  Fig. 4. Detection specificity of SADS-CoV, TGEV, PEDV, PDCoV, and PRV-A by the DPO system-based multiplex RT-PCR. a A single pair of the DPO primer was used to amplify the mixture of the five plasmid templates. (Lanes 1–6: SADS-CoV, TGEV, PEDV, PDCoV, PRV-A, and negative control). b The mixture of the five DPO primers was used with each of the five plasmid templates individually, for amplification (Lanes 1–6: SADS-CoV, TGEV, PEDV, PDCoV, PRV-A, and negative control). c Detection of the other diarrhea viruses (Lanes 1–9: positive control (P), PRRSV, APPV, SVV, PCV3, PRCV, PTV, PPV, and negative template)

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  Fig. 5. Detection sensitivity of SADS-CoV, TGEV, PEDV, PDCoV, and PRV-A by the DPO system-based multiplex RT-PCR. a Standard plasmids of TGEV, PEDV, PDCoV, SADS-CoV, and PRV-A were diluted tenfold to a final concentration between 108 copies/μL and 101 copies/μL. The cDNAs were amplified with specific DPO primer sets. b The standard plasmids of each virus were incorporated after dilution and then added to a tube containing the DPO primer mixture for PCR amplification