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ISSN : 1225-3480
본 연구는 중요한 수산자원인 전복류 중 말전복의 종-특이적 프라이머를 제작하여, PCR 분석만으로 신속하고 정확하게 말전복을 구별할 수 있는 분석 방법을 개발하였다. 약 680 bp의 미토콘드리아 DN5 유전자 분석을 통하여 종 특이성을 나타내는 단일염기다형성 유전자를 탐색하였으며, PCR 증폭산물의 크기를 고려하여 주요 전복류 4종 및 말전복의 구별이 가능한 2개의 프라이머를 제작하였다. 둥근전복, 왕전복, 북방전복은 유전적 거리가 가까워 염기서열에서 종간 특이적 차이는 발견되지 않았지만, 말전복의 경우 둥근전복, 왕전복, 북방전복간에 비해 비교적 유전적 거리가 멀어 말전복 특이적 SNP 위치를 탐색하여 종-특이적 프라이머를 제작하였다. 단일 PCR을 이용한 종간 교차반응을 통하여 최적의 PCR 조건을 확립하였으며, 증폭된 PCR 산물은 전기영동을 통해 크기에 따라 주요 전복류 4종에서는 1,006 bp, 말전복에서는 786 bp로 분리되어 명확하게 판별이 가능하였다. 말전복 종-특이적 마커의 PCR 민감도 측정에서는 <TEX>$1ng/{\mu}l$</TEX>의 농도까지 검출 가능함을 확인하였다. 따라서, 본 연구에서 개발된 전복류 4종과 말전복에 대한 종-특이적 PCR 분석법은 정확도와 민감도가 우수하여, 종 및 원산지를 속여 판매하는 불법유통 등의 수산물의 안전성 확보 및 유통질서 확립과 전복 품종간 교잡 생산을 위한 말전복 구별 등의 자원 관리 연구에 유용하게 활용될 것이라 사료된다.
This study was performed to identify rapidly Haliotis gigantea using polymerase chain reaction with species-specific primers. Around 680 bp of the mitochondrial ND5 gene region from four Haliotis species were aligned and species-specific forward primer was designed based on the single nucleotide polymorphism (SNP) from H. gigantea. The optimal PCR conditions were selected by cross reactivity using gradient PCR method from 55°C to 66°C. Species-specific PCR (SS-PCR) amplification reactions with two pairs of primers were performed for a five specimens of Haliotis species. SS-PCR leads to a species specific amplification of a 1,006 bp fragment in H. discus hannai, H. discus discus, H. madaka and 786 bp in H. gigantea, respectively. The two different sizes of each PCR products can be quickly and easily detected by single gel electrophoresis. The sensitivity of the SS-PCR was up to 1ng/μl DNA as a starting concentration in H. gigantea tested. Therefore SS-PCR technique with species-specific primer based on SNP could be a powerful tool for discrimination of H. gigantean and can contribute to the prevention of falsified labeling of this species.
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