Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
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Development and Validation of Multiplex Polymerase Chain Reaction to Determine Squid Species Based on 16s rRNA Gene

오징어류 종 판별을 위한 다중 유전자 검사법 개발 및 검증

  • Kim, Hyunsu (Department of Microbiology, Pukyong National University) ;
  • Seo, Yong Bae (Department of Microbiology, Pukyong National University) ;
  • Choi, Seong-Seok (Department of Microbiology, Pukyong National University) ;
  • Kim, Jin-Hee (Department of Food Science & Technology, Pukyong National University) ;
  • Shin, Jiyoung (Department of Food Science & Technology, Pukyong National University) ;
  • Yang, Ji-Young (Department of Food Science & Technology, Pukyong National University) ;
  • Kim, Gun-Do (Department of Microbiology, Pukyong National University)
  • Received : 2014.09.05
  • Accepted : 2014.11.12
  • Published : 2015.03.30
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Abstract

In this study, single PCR and multiplex PCR tests were examined for identification of four types of squid species (giant squid, cuttlefish, octopus, beka squid) purchased from fish market as well as aquatic processed products in Busan. To design the specific primers against each species, the nucleotide sequences of the mitochondrial 16s rRNA gene of Architeuthis dux, Todarodes pacificus, Enteroctopus dofleini, Enteroctopus megalocyathus, Uroteuthis chinensis, Uroteuthis duvauceli, Uroteuthis edulis groups were analyzed for the identification of each species registered in the GeneBank (www.ncbi.nlm.nih.gov) and have been used for comparative analysis. In order to obtain the size variation of amplified fragments on multiplex PCR, we designed KOJ-F, OJ-F, OCT-F, HAN-F, ALLR primers for each species. The optimal PCR conditions and primers were selected for four types of squid species to determine target base sequences in its PCR products. In the case of single PCR, giant squid was only amplified by KOJ-F/ALLR primer; cuttlefish was only amplified by OJ-F/ALLR primer; octopus was only amplified by OCT-F/ALLR primer; and beka squid was only amplified by HAN-F/ALLR primer. For multiplex PCR, the mixture of four kinds of genomic DNA (giant squid, cuttlefish, octopus, beka squid) been prepared as a template and used together with the mixture of KOJ-F/OJ-F/OCT-F/HAN-F/ALLR primers in the reaction. By the multiplex PCR, it is confirmed that four samples are correspond to multiple simultaneous amplicon. Finally, we validated the established methods of multiplex PCR in the aquatic processed products. Although the mitochondrial 16s rRNA primers used in this study was useful as a marker for detection of each species among them, the study indicated that the established multiplex PCR method can be more useful tool for monitoring the processed products.

본 연구에서는 오징어류에 해당하는 대왕오징어, 오징어, 문어, 한치 및 이를 이용한 가공식품에 대해서 분자생물학적 기법을 활용한 시험법을 검토하였다. 시료 중 원료성분 확인을 위하여 오징어류 4종에 대해 최적의 종 특이 프라이머를 디자인하였으며, 시료로부터 직접 genomic DNA를 추출하여 PCR을 실시하였다. PCR 수행과정에서 반응을 저해하는 염 성분을 제거하기 위하여 증류수를 이용하여 3~4회 세척 후 PCR을 실시한 결과, Single PCR의 경우 대왕오징어(552 bp), 오징어(463 bp), 문어(247 bp), 한치(354 bp)에 해당하는 종 특이적인 증폭산물을 확인하였으며, Multiplex PCR 의 경우 서로 다른 종 사이의 교차반응없이 동시다발적으로 증폭이 일어남을 확인할 수 있었다. 또한 이들 4종에 대해 PCR 민감도를 조사한 결과, 모두 약 $0.1ng/{\mu}l$의 농도까지 검출이 가능함을 확인하였으며 multiplex PCR의 경우 약 $0.25ng/{\mu}l$의 농도까지 검출이 가능함을 확인하였다. 이를 이용하여 오징어류가 함유된 수산물 가공식품 8건에 대해 적용성을 검토한 결과, 모든 시료에서 유효한 결과를 확인할 수 있었다. 따라서 본 연구에서 제작된 오징어류 4종에 대한 종 특이적 프라이머는 생물 상태뿐만 아니라 수산물 가공식품에 대해서도 이를 판별할 수 있어 식품안전관리에 활용할 수 있을 것으로 기대된다.

Keywords

References

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