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

The increasing virulence of field strains of Marek’s disease virus (MDV), which can overcome the immunity conferred by the widely used CVI988 vaccine, underscores the need for more effective alternatives. The SC9-1 vaccine strain has demonstrated superior protective efficacy and is being increasingly adopted in China. Importantly, it harbors a unique recombinant REV-LTR insertion junction that is absent from all other MDV strains. Targeting this strain-exclusive genomic locus, this study established an SYBR Green I-based quantitative real-time PCR assay for specific detection and quantification of SC9-1 to support clinical vaccine surveillance. We designed specific primers and systematically optimized the qPCR reaction conditions. The resulting assay demonstrated high specificity, showing no cross-reactivity with other MDV strains or common avian pathogens. It achieved a sensitivity of 1.0 × 101 copies/μL, and reproducibility was excellent, with both intra- and inter-assay coefficients of variation below 1.0%. Furthermore, the LOD of this assay for commercial SC9-1 vaccine samples was determined to be 1.22 PFU per 200 μL sample. In conclusion, this SYBR Green I-based qPCR assay provides a specific, sensitive, and reproducible tool for the rapid quantification of the SC9-1 vaccine virus. It is fully validated for routine vaccine quality control, while exhibiting promising potential for subsequent field surveillance within MD vaccination programs.