Validation of mitochondrial COX3 primers for quantitative PCR-based authentication of chicken-derived products

This study developed and validated a novel, highly specific primer set targeting the mitochondrial cytochrome c oxidase subunit III (COX3) gene for the detection of chicken DNA (Gallus gallus domesticus) using quantitative PCR (qPCR) to enhance food authentication reliability. A 70 bp chicken-specific amplicon was designed and validated for repeatability, analytical specificity across six animal species, and sensitivity. The assay demonstrated high precision, with an average quantification cycle (C_q) of 13.18 ± 0.20 and a stable melting temperature (T_m) of 81.27 ± 0.05 °C (RSDr <1.5%). Analytical specificity tests confirmed the detection of chicken DNA with no cross-amplification in non-target species, including beef, pork, sheep, squid, shrimp, and ducks. The absolute functional limit of detection (LoD) was 10.09 pg/μL, while the relative LoD in binary DNA mixtures reached 0.5% (v/v) with 100% PCR efficiency and R² = 0.999. Application of this assay to 17 commercial meat products (sausages and floss) revealed a high incidence of mislabeling, where undeclared chicken DNA was detected in seven out of 11 samples (63.6%) declared as 100% beef, representing 41.2% of the total samples tested. These findings indicate that the COX3 primer set provides an accurate and efficient approach for authenticating animal-derived ingredients, significantly enhancing consumer protection against food adulteration.