Application of COI barcode sequence for the identification of snake medicine (Zaocys)

Abstract

Counterfeits in the medicine market make the authentication of snakes used for Chinese medicine a challenge to Chinese drug regulatory control agencies. This paper explores existing methods that can be used to quickly and accurately distinguish Zaocys (Z. dhumnades) from its counterfeits for routine identification of snake meats in food and drug control laboratories. In this research, the Cytochrome Oxidase I (COI) fragments of 51 samples from 17 species of snakes were amplified using Polymerase Chain Reaction (PCR) and sequenced. The inter- and intra-specific variations of COI sequences were analyzed and compared based on Kimura-2-parameter (K-2P) distances; the minimal interspecific K-2P distance was 0.0934, which was bigger than the maximum intraspecific K-2P distance in Z. dhumnades (0.0523), indicating that Zaocys can be separated from its counterfeits. The Neighbor-Joining (N-J) tree of the snakes was constructed and the results show that snakes of the same species cluster with 100% bootstrap values. Since the Zaocys and its counterfeits are of different species, they can be distinguished using the N-J tree method. Another 10 samples of Zaocys from markets and drug stores were identified at the species level, among which 5 samples were proven to be the counterfeits--Ptyas korros.

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

“…In the present study, considerable fresh Shedan of original animals identi ed by morphology were collected. The previous studies had reported that COI gene universal primers (LCO1490/HCO2198) were appropriate for distinguishing snake species from the families of Colubridae and Elapidae [19,20,22]. Therefore, COISNFF/COISNFR primers were optimized based on LCO1490/HCO2198 primers and their corresponding region in COI gene of snakes by this study.…”

“…Thereinto, a fragment (~650 bp) of the barcode region in mitochondrial cytochrome c oxidase submit I (COI) gene, has been used as the most effective DNA barcode marker for identifying and classifying animal-derived medicinal materials even with highly similar or incomplete morphological traits [15][16][17][18]. Previous studies have displayed that DNA barcoding can accurately identify different snake species and distinguish snake-related medicinal materials from adulterants and substitutes through amplifying the COI sequence of the muscle tissue or periostracum serpentis [19][20][21][22][23][24], and none of these studies focused on snake gallbladder or snake bile. It was worth noting that the ampli cation primers of these studies were incompletely identical, but COI gene universal primers (LCO1490/HCO2198) [25] were mostly adopted.…”

Application of Molecular Methods in the Identification of Ingredients in Chinese Herbal Medicines

“…Nowadays, this technology has been applied widely, and can be regarded as a mature tool for biological exploration and research. Not only can it be used to identify organisms that are difficult to distinguish by other methods, it can also be used to discover new organisms that have not been discovered by the biological research community to date [ 12 ]. If a specimen of a suspected new organism is collected, this technique can be used to extract its corresponding DNA barcode, and then compare and analyze it with DNA barcodes (nucleotide sequences) that are available in the public nucleotide sequence database, so that the relevant identity information of this organism can be determined step-by-step.…”

Phylogeography of the reticulated python (Malayopython reticulatus ssp.): Conservation implications for the worlds’ most traded snake species

“…They have been used effectively during wildlife forensic investigations (see [ 42 ] for review)—to identify parts of animal derivatives in an otherwise unidentifiable sample. Forensic procedures have been developed to identify animal parts or derivatives in the traditional Chinese medicine trade (e.g., snakes: [ 43 , 44 ]), identify turtle species from samples of shell [ 45 ], to verify origin [ 46 ] either where illegal activity or fraudulent claims are suspected e.g., Testudo graeca [ 47 ] and Morelia boeleni [ 48 ], and to authenticate population of origin where regional differences in population viability, management or harvest quotas exist (e.g., ivory, [ 49 ]; shark fins, [ 50 ]; regulated fisheries, [ 51 ]). Taxonomic clarification prior to monitoring trade was suggested for short-tailed pythons, the polytypic species Python curtus [ 52 ].…”