太平洋褶柔鱼及南海鸢乌贼双位点特异性PCR鉴定方法的建立

A double site-specific PCR system for authentication of Todarodes pacificus and its adulterant Sthenoteuthis oualaniensis

目的:市场上利用南海鸢乌贼冒充太平洋褶柔鱼作为鱿鱼加工原料的现象时有发生,拟为2种鱿鱼建立一种简单高效的DNA分子鉴定方法。方法:利用从线粒体细胞色素氧化酶亚基I(COI)发掘的太平洋褶柔鱼和南海鸢乌贼的SNP位点,分别设计2种鱿鱼的双位点特异性引物,利用多重PCR对太平洋褶柔鱼和南海鸢乌贼进行鉴定和区分。结果:双位点特异性引物在相对温和的退火温度下对2种鱿鱼品种显示了绝对的特异性,太平洋褶柔鱼和南海鸢乌贼分别扩增出了193bp和530 bp的品种特异性条带。建立的多重PCR体系可以有效地对太平洋褶柔鱼和南海鸢乌贼及两者的混合品进行鉴定。结论:多重PCR方法为市场上太平洋褶柔鱼和南海鸢乌贼的鉴定提供了有效的DNA分子手段,并可为太平洋褶柔鱼及其加工产品的原料来源检测提供客观依据。

Todarodes pacificus and Sthenoteuthis oualaniensis are two different squid species in the market. Due to their differences in texture, nutrition value, and price, S.oualaniensis is often found to be adulterants of T. pacificus. The aim of this study is to develop a simple and effective method for molecular authentication of T. pacificus and S.oualaniensis. Methods： Based on single nucleotide polymorphisms（SNP） exploited from mitochondrial cytochrome oxidase subunit I（COI） region, two double site specific primer pairs were designed for T. pacificus and S.oualaniensis by introducing additional mismatches both in the forward and reverse primers. Multiplex polymerase chain reaction（PCR） was conducted for identification of T. pacificus and S.oualaniensis. Results： The developed double site specific primer pairs showed absolute specificity despite moderate annealing temperature, T. pacificus and S.oualaniensis generated their specific amplicons of 193 bp and 530 bp, respectively. The established multiplex PCR method is effective for authentication of T. pacificus and S.oualaniensis as well as their mixture. Conclusion： The present study provides an effective method for molecular identification for T. pacificus and S.oualaniensis in the market, and it can be used as an objective criteria for quality control of squid products.