A calibration-enhanced real-time PCR method for bovine ingredient quantification using single-copy nuclear gene markers

• A novel quantitative real-time PCR (qPCR) assay was developed for the specific detection and accurate quantification of bovine-derived ingredients in complex, processed meat products. • The method targets a non-coding region of the single-copy phosphodiesterase (PDE) gene from the bovine nuclear genome as the target gene (beef) and the myostatin gene, which is widely present in vertebrates, as the reference gene (ref), overcoming limitations associated with variable mitochondrial DNA copy numbers used in traditional methods. • A key innovation is the introduction of a calibration factor (k) derived from digital PCR (dPCR) absolute quantification, which corrects for systematic errors in DNA extraction and amplification efficiency, ensuring high accuracy. • The assay demonstrates exceptional analytical performance: a low limit of detection (LOD of 10 copies/μL), a limit of quantification (LOQ of 100 copies/μL), high trueness (4.78%), and excellent precision (repeatability RSD of 9.62%). • The method was rigorously validated according to international guidelines and successfully applied to analyze 30 commercial products, uncovering significant mislabeling and adulteration in steak, meatball, and jerky samples. • This work provides a robust, reliable, and highly accurate tool for regulatory agencies and food control laboratories to authenticate beef products, combat food fraud, and protect consumer rights. Quantitative analysis of bovine-derived components is essential for combating meat adulteration in processed foods, yet accurate quantification remains challenging. This study developed a reliable qPCR method targeting the single-copy nuclear cyclic GMP phosphodiesterase ( PDE) gene (bovine-specific) and the myostatin gene (universal reference). A correction factor was introduced to minimize systematic errors, enabling precise cellular percentage quantification. Results are expressed as cellular percentage, avoiding mass fraction inaccuracies. The method demonstrated high sensitivity (LOD: 10 copies/µL; LOQ: 100 copies/µL), accuracy (trueness: 4.78%), and precision (RSD: 9.62–17.60%), with excellent linearity (R² = 0.9981) from 10² to 10⁸ copies/µL. Application to 30 commercial samples revealed significant adulteration: beef contents below 50% in steak and beef granules, no bovine material in beef balls, and a suspect beef roll sample with 73.09% content.