大西洋三文鱼和虹鳟鱼双重PCR检测方法的建立及应用

Development and Validation of a Duplex PCR for the Simultaneous Detection of Atlantic Salmon (Salmo salar) and Rainbow Trout (Oncorhynchus mykiss) in Processed Fish Products

摘要:

为了实现大西洋三文鱼和虹鳟鱼的物种掺假快速检测，本研究建立了一种基于双重PCR（普通和荧光）的物种鉴定方法。通过序列分析，分别基于线粒体COⅠ和Cyt b基因设计大西洋三文鱼和虹鳟鱼特异性引物，验证引物的特异性并对双重PCR反应体系进行优化，建立大西洋三文鱼和虹鳟鱼的双重PCR快速鉴定方法。利用本研究设计的引物，仅大西洋三文鱼和虹鳟鱼可以分别扩增出108 bp和207 bp的特异性条带，而其他23种非目标物种均未有扩增条带。经验证，在双重PCR反应体系中，大西洋三文鱼和虹鳟鱼引物的最佳添加量分别为0.1和0.4 μmol/L，熔解温度分别约为81.5 ℃和85 ℃。利用该方法从29份市售“三文鱼”样品中检测到6份大西洋三文鱼和3份虹鳟鱼，且与DNA条形码比对结果一致。本研究建立的大西洋三文鱼和虹鳟鱼双重PCR（普通和荧光）鉴定方法具有良好的特异性和适用性，为大西洋三文鱼和虹鳟鱼物种鉴定提供了可靠的技术手段。

Abstract:

Anovel method for rapid identification of Salmo salar and Oncorhynchus mykiss based on duplex PCR assay (conventional and fluorescent assays) was developed. Specifically, after sequence alignment, specific primers for S. salar and O. mykiss based on the COⅠ and Cyt b genes were designed and verified. The duplex PCR was optimized, and finally established. Only S. salar and O. mykiss amplified specific bands of 108 bp and 207 bp, while the other 23 non-target species were not successfully amplified. The optimal amount of primers for S. salar and O. mykiss were 0.1 μmol/L and 0.4 μmol/L, and the melting temperature was 81.5 ℃ and 85 ℃, respectively. Among the 29 commercial products, six products were identified as S. salar and three were identified as O. mykiss. Finally, theremaining 17 commercial samples were all tested as negative in the duplex PCR assay, and the results were consistent with DNA barcoding method. The results showed that the duplex PCR method for S. salar and O. mykiss was specific and feasible. This study provided reliable technical method and theoretical basis for the adulteration identification of S. salar and O. mykiss species. Key words: