Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Development of the Duplex PCR Method of Identifying Trachurus japonicus and Trachurus novaezelandiae

다중 PCR 분석법을 이용한 전갱이속 어종의 신속한 종판별 분석법 개발

  • Park, Yeon Jung (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Lee, Mi Nan (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Kim, Eun Mi (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Noh, Eun Soo (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Noh, Jae Koo (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Park, Jung Youn (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Kang, Jung-Ha (Biotechnology Research Division, National Institute of Fisheries Science)
  • 박연정 (국립수산과학원 생명공학과) ;
  • 이미난 (국립수산과학원 생명공학과) ;
  • 김은미 (국립수산과학원 생명공학과) ;
  • 노은수 (국립수산과학원 생명공학과) ;
  • 노재구 (국립수산과학원 생명공학과) ;
  • 박중연 (국립수산과학원 생명공학과) ;
  • 강정하 (국립수산과학원 생명공학과)
  • Received : 2018.05.09
  • Accepted : 2018.08.27
  • Published : 2018.09.30
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Abstract

Reliable labeling of fish products can reassure consumers regarding the identity and quality of seafoods. Therefore, techniques that can identify adulteration or mislabeling are valuable. To rapidly identify two Trachurus species, Trachurus japonicus and Trachurus novaezelandiae, a highly efficient, rapid, duplex polymerase chain reaction (PCR) having two species-specific primers simultaneously was identified. This species-specific primer focused on a single nucleotide mismatch in the 3'-terminal base of a primer designed in the mitochondrial cytochrome c oxidase (COI) subunit I DNA. To optimize the duplex PCR condition, gradient PCR reactions were conducted to determine the primer annealing temperature and the primer concentration. The PCR's product was observed on the gel, suggesting that DNA molecules may be useful in differentiating the two species. The length of the amplification fragments were 103 bp for Trachurus japonicus and 214 bp for Trachurus novaezelandiae, which, along with the species-specific primer visualized by agarose gel electrophoresis, enabled accurate distinction of the species of the Trachurus genus. These results indicate that the duplex PCR, which has a species-specific primer based on single nucleotide polymorphism (SNP), can be useful for rapidly differentiating the two species of Trachurus. This duplex PCR analysis is simple, rapid, and reliable, and could be beneficial to protecting consumers' rights.

국제 무역 및 전세계 수산물 소비의 증가로 인해 다양한 수산물이 국내로 수입되어 유통되고 있다. 최근 수입 수산물의 종명 및 원산지 표시사항을 허위로 기재하는 경우가 급증하여 식품안전성에 심각한 문제가 야기되고 있다. 불법적으로 유통되는 수산물의 안전관리를 위해 DNA 기반 기술을 이용한 종 판별법 마련이 시급하다. 본 연구에서는 전세계적으로 중요한 대형선망어업 어종 중 하나인 전갱이속 어류의 종을 판별하기 위해 duplex-PCR을 사용한 검출 방법을 개발하였다. 국내에 유통되는 T. japonicus과 T. novaezelandiae의 시료를 확보하여 COI 영역의 염기서열 분석을 통하여 종간 특이성을 나타내는 단일염기다형성 유전자를 탐색하였으며, PCR 증폭 산물의 크기를 고려하여 2개의 종 특이적인 정방향 primer를 설계하였다. Duplex-PCR 분석 결과, T. japonicus (103 bp), T. novaezelandiae (214 bp)와 같은 단일 밴드를 전기영동상에서 확인 할 수 있었으며 상호간의 비 특이적 밴드는 형성되지 않았다. 또한 duplex-PCR 방법을 통한 T. japonicus과 T. novaezelandiae에서 최저 $0.01ng/{\mu}l$까지 검출됨을 확인 할 수 있었다. 따라서 본 연구에서 개발된 duplex-PCR 분석법을 이용한 전갱이속 어류의 종 판별법은 정확도와 민감도가 우수하여 수산물의 수출입 및 시중에 불법적으로 유통 가능성이 있는 제품을 신속하고 과학적으로 판별할 수 있어 수산물안전관리에 활용도가 매우 클 것으로 기대된다.

Keywords

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