Optimization and validation of the real-time polymerase chain reaction protocol for porcine gelatin detection in processed food and cosmetic products

Background: Processed food and cosmetic products are integral to modern life, and technological advances have enabled the creation of increasingly complex formulations. Many of these products contain animal-derived ingredients, such as gelatin, whose source must be verified due to religious, health, and ethical concerns. Porcine is one of the primary sources of gelatin production, which has become a major concern. Its detection in complex matrices, such as processed food and cosmetics is challenging, as gelatin itself undergoes extensive production processes. Real-time polymerase chain reaction (PCR) is widely recognized as one of the analytical methods of choice for species determination; however, its application to gelatin source identification in complex samples remains limited. Aim: This study aimed to develop and optimize a real-time PCR protocol for identifying porcine gelatin contamination in highly processed matrix samples (processed foods and cosmetics) for halal authentication. Methods: This study was established through the following steps: DNA extraction, post-isolation DNA analysis, annealing temperature and primer concentration optimization, specificity assay, amplification efficiency trial, sensitivity test, repeatability examination, and marketed sample analysis. Results: The developed method demonstrated good specificity under optimized conditions (annealing temperature of 58.4 °C and primer concentration of 0.2 µM). It achieved a good amplification efficiency of 101.2% with an R² of 0.994. Further validation results showed that the real-time PCR technique had a limit of detection of 1,316 pg in the sensitivity examination and a satisfying coefficient of variation of 0.81% in the repeatability testing. The marketed sample analysis (utilizing five cosmetic samples in the form of facial masks and five food samples in the form of one food additive gelatin powder, two marshmallow products, and two gummy candy products) showed that all of the samples displayed no amplification and were thus considered not to contain porcine DNA, consistent with the manufacturers’ labels. Conclusion: The real-time PCR method meets the validation criteria for qualitative analysis, including specificity, amplification efficiency, sensitivity, and repeatability. This method provides a valuable foundation for advancing halal authentication, particularly for detecting porcine gelatin contamination in processed food and cosmetic products.