Application of DNA Barcoding for the Identification of Snake Gallbladders as a Traditional Chinese Medicine

Snake gallbladders, or shedan, have a long history of application in Traditional Chinese Medicine. However, different sources have been indiscriminately used due to the lack of a robust methodology to identify and authenticate the crude drug, causing great risk to the safety and effectiveness of clinical applications. Hence, a practicable approach to identifying snake gallbladder crude drugs is indispensable, and its development is described here. Folmer primers and novel COISNF primers were separately evaluated to screen the more specific primers for the investigation of 253 snake gallbladder samples from 31 species, 51 snake bile samples from 17 species, 17 bile samples from five common adulterant species (cattle, chicken, duck, pig, and sheep), and 195 snake gallbladder samples from 18 batches of commercial crude drugs. Sequence definition was executed by querying similarities in GenBank and the Barcode of Life Data System. COISNF primers, rather than Folmer primers, could distinguish the tested snake gallbladder samples from each species, and COISNF primers were specific to snake species and the other four adulterant animal species except duck. In commercial samples, 13 snake species and adulterant chicken species were found. The original source of current commercial snake gallbladder samples has been preliminarily clarified, laying the groundwork for the quality control of snake gallbladder crude drugs. Graphical abstract