Identification of Turtle Shell and Its Preparations by PCR-DNA Sequencing Method   全文下載

Identification of Turtle Shell and Its Preparations by PCR-DNA Sequencing Method

A polymerase chain reaction (PCR) with DNA sequencing method has developed to identify the species of turtle shell specimens, skinned and bleached turtle shells, and its preparations. Two pairs of universal primer of mitochondrial gene, 12S rRNA (L:1373, H:1478) and cytochrome b (L:14181, H:15149), were applied to perform PCR and test for the optimal PCR conditions. The PCR products of about 165-bp and 376-bp in size were obtained from all 12 authentic turtle shells by primers of the 12S rRNA gene and the cytochrome b gene, respectively. Following DNA sequencing analysis of both bands without primers and GeneBank database search, DNA sequences of about 108-bp of 12S rRNA fragments were well differentiated among the 12 authentic specimens. However, DNA sequences of 307-bp of cytochrome b fragments were not completely distinguished for those authentic specimens. The PCR method with the primers of mitochondrial 12S-rRNA can also be successfully applied to the skinned and bleached turtle shell, and its powder preparation to identify their corresponding species. Thirty samples of skinned and bleached turtle shells were identified and categorized as three different species of Siebenrockiella crassicollis, Indotestudo elongata, and Cuora amboinensis, respectively. Furthermore, one extract and three powders of turtle shell preparations were identified as Siebenrockiella crassicollis, and one of turtle extract preparations was shown the cross-amplification with the other animal. Turtle jelly preparation, however, did unidentifiable. The results also indicated that the endangered species of turtle listed in CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) Appendix I and the prohibition of wild fauna announced by the Committee on Chinese Medicine and Pharmacy, Department of Health, Taiwan were not used as raw materials in the Chinese pharmaceutical manufacturers.