A high-sensitivity qPCR method for detecting residual Vero cell DNA in rabies vaccine production.

Residual host cell DNA in biological products requires precise quantification to meet regulatory safety standards. This study presents the development and validation of a qPCR assay for detecting and quantifying residual Vero cell DNA in rabies vaccine formulations. A SYBR Green-based quantitative PCR (qPCR) assay targeting the Vero cell-specific alpha-satellite DNA sequence was developed and validated in accordance with regulatory guidelines. The method was evaluated for specificity, sensitivity, linearity, precision, and robustness using a certified genomic DNA standard. The assay exhibited high specificity with no amplification from non-target DNA. The method demonstrated a linear dynamic range from 0.064 to 1000 ng/mL (R² > 0.999) with an amplification efficiency of 96.3 %. Sensitivity analysis confirmed reliable detection well below the regulatory threshold of 10 ng per dose, with a calculated limit of quantification of 0.31 ng/mL. Intra- and inter-assay precision tests showed low variability (CV < 20 %), supporting assay reproducibility. Accuracy was confirmed through recovery experiments (93.33-117.33 %) and concordance with a reference method (kappa = 1). This validated qPCR method offers a sensitive, specific, and reproducible tool for monitoring residual Vero cell DNA in rabies vaccine production, ensuring regulatory compliance through robust quality control.