Development of a species-specific polymerase chain reaction-based technology for authentication of asini corii colla and taurus corii colla

Background: Asini Corii Colla (ACC; donkey-hide glue) and Taurus Corii Colla (TCC; bovine-hide glue) are popular healthcare food supplements as well as well-recognized medicine in Traditional Chinese medicine clinics. Due to large demand of them and limited resources of their animal origins, the skin of other animals are often used to make their fake or adulterated products. Objective: In this study, we aimed to develop a species-specific polymerase chain reaction (PCR) approach to detect deoxyribonucleic acid (DNA) fragments of ACC and TCC for rapid authentication purpose. Materials and Methods: Four sets of novel species-specific primer were designed, and PCR conditions were optimized for the PCR assay, which was further validated for specificity and sensitivity. Then, deliberate mixture was analyzed to further verify the capability of adulteration detection by the developed PCR method. Finally, it was used to assess the authenticity of commercial products. Results: Four primers' sets were specific for amplification of extracted mitochondrial DNA of donkey, bovine, swine, and horse when the annealing temperature was 59°C, 59°C, 63°C, and 65°C, and 40 thermal cycles was performed. Under this optimized PCR conditions, 1 ng/μL of horse DNA template has been detected in the sensitivity test, with 0.1 ng/μL for donkey, bovine, and swine. The assay was capable of detecting spiked species at 10% level in premixed ACC or TCC samples. Conclusion: The newly developed PCR-based technology was specific and sensitive, and it was a convenient approach for the authentication of commercial ACC and TCC products.