The Development and Evaluation of an SYBR Green I-Based qPCR Assay for Detecting the Marek’s Disease Virus SC9-1 Vaccine Strain

Figures

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  Figure 1. Optimization of annealing temperature for the qPCR thermal cycling protocol. The results indicate that (a) the amplification curve achieved the strongest fluorescence intensity and the lowest cycle threshold value at an annealing temperature of 60.1 °C. (b) The corresponding melting curve presents a single distinct amplification peak.

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  Figure 2. Gradient screening of primer concentrations for qPCR. (a) A primer concentration of 0.4 μM yielded high fluorescence intensity and low Ct values; further concentration elevation (0.5–0.6 μM) did not improve amplification performance. (b) All tested concentrations generated single melting peaks with no detectable primer dimers.

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  Figure 3. The construction and linear validation of the recombinant plasmid standard. (a) A specific 101 bp target fragment was amplified from SC9-1 vaccine DNA, purified, cloned into the pCE2-T/A vector and transformed into E. coli DH5α. (b) Serial 10-fold dilutions (1.0 × 101–1.0 × 109 copies/μL) of purified plasmid were used to generate a standard curve. The linear regression equation was y = −3.462x + 39.616, with R2 = 0.999 and an amplification efficiency of 94.5%.

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  Figure 4. Specificity analysis of the SYBR Green I-based qPCR assay. The established assay specifically amplified DNA from the SC9-1 vaccine strain (curve 1) and its corresponding recombinant plasmid standard (curve 2). No amplification signals were detected from nucleic acids of other MDV strains or common avian pathogens (curves 3–19: CVI988, 814, Md5, HVT, WTMDV-1~WTMDV-7, CIAV, ALV-J, REV, IBDV, NDV and NTC).

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  Figure 5. Sensitivity detection of the qPCR assay using serially diluted recombinant plasmid. Curves 1–9 correspond to plasmid concentrations from 1.0 × 101 to 1.0 × 109 copies/μL. The limit of detection (LOD) of this method was determined as 1.0 × 101 copies/μL.

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  Figure 6. LOD evaluation using serially diluted commercial SC9-1 vaccine samples. Curves 1–6 represent vaccine nucleic acids ranging from 1.22 × 106 PFU/200 μL to 1.22 PFU/200 μL, with Ct values of 14.08, 18.12, 23.14, 28.48, 33.41 and 37.14, respectively. No target amplification was detected at 0.122 PFU/200 μL (curve 7), consistent with the NTC.