一种基于荧光PCR法检测虹鳟鱼源性成分方法的建立

Detection of Rainbow Trout Ingredients by Fluorescence-Based Quantitative Real-Time PCR

目的:建立一种基于荧光聚合酶链式反应技术鉴别虹鳟鱼的检测方法。方法:以细胞色素C氧化酶Ⅰ为目的基因,设计特异性引物和探针,通过测试引物特异性和模拟检测虹鳟鱼与三文鱼鱼肉混合物验证该体系的检测效果。结果:该虹鳟鱼荧光聚合酶链式反应检测体系具有很好的特异性及灵敏性,可特异性地检测出含1%(w/w)虹鳟鱼的DNA存在。说明该体系抗三文鱼的干扰性强,可在1 h内稳定地检测出虹鳟鱼源性成分。结论:该方法检测效果好、时间短、成本低,可为监管部门解决市场中虹鳟鱼源性成分的监管问题提供技术手段。

Objective: This study is aimed to establish a fluorescence-based real time polymerase chain reaction(PCR) assay for specific detection of rainbow trout ingredients. Methods: A pair of primers and Taq Man probe targeting the conservative regions of the rainbow trout mitochondrial cytochrome oxidase subunit Ⅰ(COⅠ) gene were designed. The assay system was validated with specificity by detecting several seafood and sensitivity by detecting salmon with rainbow trout mixed meat. Results: The assay was found to be highly specific and sensitive as evidenced by showing no amplification of DNA extracted from other seafood and its capability of detecting DNA from 1%(w/w) rainbow trout meat. From those results we can confirm that the assay system can anti-interference rainbow trout meat strongly and the whole test could be finished within 1 hour. Conclusion: The PCR system is an effective method as well as rapidity and inexpensive and can provide a technical means for market regulation to detect rainbow trout ingredients.