Application of DNA Barcoding for the Identification of a Traditional Chinese Medicine Shedan

Background Shedan has a long history of application in Traditional Chinese medicine (TCM), however, Shedan from different original source has been indiscriminately used. So far, there is still a lack of an effective tool to differentiate the original source of Shedan medicinal materials, which brings great risk to the safety and effectiveness of clinical applications. Hence, it is imperative to develop a practicable approach to identify Shedan medicinal materials. Methods The specificity of two pairs of primers, including Folmer’s universal primers and a pair of originally designed primers COISNFF/COISNFR, was tested to screen the more specific primers for further origin identification of Shedan. A total of 253 fresh snake gallbladder samples from 31 morphologically identified snake species were collected and authenticated. Moreover, 51 fresh snake bile samples and 17 fresh bile samples from five other common domestic poultry and livestock (cattle, chicken, duck, pig and sheep) were collected and distinguished using the more specific primers. Additionally, a total of 195 market Shedan samples randomly selected from 18 batches of Shedan medicinal materials were investigated. Sequence definition was executed by querying sequence similarities in GenBank and the Barcode of Life Data System (BOLD), respectively. Results It turned out that the standard COI barcode obtained by COISNFF/COISNFR primers, rather than Folmer’s universal primers, can distinguish all the testing samples from each other in fresh Shedan samples, and COISNFF/COISNFR primers were also specific to snake species and the other four animal species except duck. In terms of market Shedan, 84.6% (165/195) samples can be attributed to 13 snake species from four families and 4.6% (9/195) can be attributed to adulterated chicken species. Conclusion The COI-based DNA barcoding was practicable for species identification of Shedan used in traditional Chinese medicine. The original source of current market Shedan, including adulterated species, has been preliminarily clarified, which provides a foundation for quality control of Shedan medicinal materials.