Development and verification of a quantitative real-time PCR method to identify and quantify gelatin derived from animal hide.

The species origin of hide gelatin is a crucial issue with respect to health concerns and religious restrictions. Analysis of the animal-derived ingredients of gelatin by reliable methods is necessary to ensure its authenticity. However, due to the highly processed nature of gelatin, it remains a challenge to identify gelatin end products accurately and robustly. Our study established and verified a quantitative real-time PCR (qPCR) method based on careful selection of target genes and a DNA extraction method. The middle products of the gelatin production streamline were investigated to explore the influence of each critical processing step on the method. Gelatin reference samples were used to quantify the levels of target species. Commercial gelatin commodities were surveyed to highlight the mislabeling situation. In summary, the qPCR method was demonstrated to be highly specific and sensitive, with limits of detection (LOD) of 0.1 to 1 pg/µL and gelatin LODs of 0.1% to 5% (w/w). The transition from decoction to concentrated gel was found to have the most severe effect on the qPCR. Intensification of pressure or temperature or employment of enzyme hydrolysis aggravated the DNA damage, resulting in elevated Cq values. Quantitation of gelatin products was feasible; gelatin products produced from 5% target hide and 95% matrix hide mixtures showed 2.9% to 5.2% target species. The 26% relative error for low gelatin content is acceptable for semiquantitation purposes. A market survey showed that 52.6% of the gelatin products were mislabeled as being of animal origin.