두족류의 진위 판별을 위한 Real-time Quantitative PCR 검사법 개발 및 검증

In this study, an approach for the analysis of the five cephalopod species (octopus, long-arm octopus, squid, wet-foot octopus, beka squid) consumed in the Republic of Korea is developed. The samples are collected from the Southeast Asian countries Thailand, Indonesia, Vietnam, and China. The SYBR-green-based real-time qPCR method, based on the mitochondrial DNA genome of the five cephalopods was applied and validated. The intergroup variations in the mitochondrial DNA are evident in the bioinformatic analysis of the mitochondrial genomic-DNA sequences of the five groups. Some of the highly-conserved and slightly-variated regions are identified in the mitochondrial cytochrome-c-oxidase subunit I (COI) gene, 16s ribosomal RNA (16s rRNA) gene, and 12s ribosomal RNA (12s rRNA) gene of these groups. To specify each of the five cephalopod groups, specific primer sets were designed from the COI, 16s rRNA and 12s rRNA regions. The specific primer sets amplified the DNA using the SYBR-green-based real-time PCR system and 11 commercially secured animal tissues: Octopus vulgaris, Octopus minor, Todarodes pacificus, Dosidicus gigas, Sepia esculenta, Amphioctopus fangsiao, Amphioctopus aegina, Amphioctopus marginatus, Loliolus beka, Loligo edulis, and Loligo chinensis. The results confirmed by a conveient way to calculate relative amplification levels between different samples in that it directly uses the threshold cycles (Ct)-value range generated by the qPCR system from these samples. This genomic-DNA-based molecular technique provides a quick, accurate, and reliable method for the taxonomic classification of the animal tissues using the real-time qPCR.