Studies on identification gene assay for shellfish and related products in Taiwan

• Main Author: Chang, Kuan-Lin
• Other Authors: 保健營養生技學系
• Format: Thesis
• Language: Chinese; English
• Published: Asia University 2014
• Subjects: shellfish; Crassostrea gigas
• Online Access: http://asiair.asia.edu.tw/ir/handle/310904400/80715; http://asiair.asia.edu.tw/ir/bitstream/310904400/80715/2/index.html

Shellfish is one of the most important fisheries resources in Taiwan. Before utilizing, most times they’re shucked, processed (freezing, or heating, etc.) and stored. Hence, as a result of losing distinguishable exterior characteristics, other identify approaches are needed for shellfish species forensics. Due to the severe processed procedure, protein is cleaved. Thus, analytic tools of proteins are not applicable to processed shellfish species. Therefore, the study adopts genetic technologies for identification. The level and differences of the storage and processing time will affect the completeness of the genome sequencing, and lead to the result of the identification. For this reason, firstly the research observes the DNA cleavage status of the sample, choosing suitable length of the target genome with related genetic technology for shellfish species identification. In Taiwan, popular fisheries and aquaculture of shellfish species include Geloina erosa, Corbicula fluminea, Meretrix lusoria, Cyclina sinensis, Mizuhopecten yessoensis, Sinonovacula constricta, Ruditapes philippinarum, Perna viridis, Tegillarca granosa, and Crassostrea gigas. The study establishes these 11 shellfishes’ complete COI genome sequencing and 9 Cyt b complete genome sequencing, which the base pairs are 1740 bp, 1,445 bp respectively. With the suitable genome base pairs data of shellfish species, the study sets it as the primer design of the species identification. Moreover, by utilizing the established COI genome, the study then designs the common but solely primer: COI-F/R, which the base pair is 460 bp. After adopting the primer with PCR-RFLP technology of 5 restriction enzymes: BsaJI、HaeIII、BmrI、MslI、NlaIII, the research successfully identifies 11 unprocessed and processed shellfish species. Since the products are often processed under circumstances like high temperature or blended with other shellfish species, the DNA completeness is influenced. The study establishes multiplex-PCR primers Geloina-F/R, Gigas-F/R, and Corbicula-F / R which the base pairs are 183 bp, 122 bp, and 256 bp respectively to identify fake clams, Geloina erosa clams and oysters clams. As for fake scallop sauce, the study utilize Cyt b genome to design the specific primer yessoensis-F/R and successfully increase the base pairs to 320 bp. According to the above testing results, the research approves the applicability of PCR-RFLP and multiplex-PCR. By collecting shellfish related samples all over Taiwan, including 33 oyster clam tab and 24 scallop sauce products, the testing results indicate that there is one unqualified product of oyster clam tab whereas the identification possibility of scallop sauce is 29.1%.