Detection of Chickpea Residue in Food by Molecular Biomarker Analysis — Development of DNA Detection Method and ISO Standardization

Chickpeas, a high-protein emerging plant-based food source, face allergenicity concerns necessitating robust residue detection in the food supply chain. This study established a DNA detection method by real-time PCR targeting the Chloroplast import apparatus 2-like gene fragment (chromosome Ca7) for detection of chickpea residue, optimized for ISO standardization. Utilizing molecular biomarker analysis, the method demonstrated an absolute limit of detection (LOD) of 5 copies and a LOD_95% of 4.08 copies, enabling detection at 0.01% (w/w) mass fraction. Robustness yielded an RSD of 0.86%. Specificity was confirmed against 49 non-target species, and inclusivity was validated across 9 chickpea varieties. International collaborative trials exhibited 0% false positive/negative rates, a LOD_95% of 3.601 copies, a probability of detection (POD) of 0.638, and an inter-laboratory standard deviation of 0.331. Successful application to real food samples confirmed applicability. This validated method, advanced to the ISO/CD TS 24910-1 stage, provides critical technical support for global food safety by detecting chickpea residues.