双重实时荧光聚合酶链反应法快速鉴定羊肉制品中鸭源性成分

Rapid identification of duck-derived components in mutton products by duplex real-time fluorescent polymerase chain reaction

中文关键词:  羊肉制品  鸭源性成分  DNA粗提  响应面法  双重实时荧光聚合酶链反应法

英文关键词:mutton products  duck-derived components  crude DNA extraction  response surface methodology  duplex real-time fluorescent polymerase chain reaction

中文摘要:

      目的 建立双重实时荧光聚合酶链反应法(duplex real-time fluorescence polymerase chain reaction, dRT-PCR)快速鉴定羊肉中鸭源性成分的方法。方法 首先使用响应面法优化快速提取动物组织DNA的各项条件, 随后根据羊线粒体细胞色素b基因(CYTB)和鸭细胞色素c氧化酶亚基Ⅲ (COX3)的差异设计多组特异性引物, 使用快速提取的动物组织DNA进行dRT-PCR实验, 通过分析熔解曲线, 根据出峰温度差异以定性识别羊、鸭源成分。结果 优化后的动物组织DNA粗提方法可在5 min内从20 mg动物组织中提取出586 ng/μL适用于物种检测所用的DNA, 羊源基因产物和鸭源基因产物分别在84.79 ℃和81.66 ℃时产生熔解峰, 且对非目标物种无特异性扩增。可在1 h内快速检出0.01 ng/μL以上鸭粗提DNA。结论 该方法操作简单, 成本低廉, 具有较高灵敏度, 为羊肉制品掺假鉴定提供了有效技术支持。

英文摘要:

      Objective To establish a method for the rapid identification of duck-derived components in mutton by duplex real-time fluorescence polymerase chain reaction (dRT-PCR). Methods First, the response surface methodology was used to optimize various conditions for the rapid extraction of DNA from animal tissues. Subsequently, multiple sets of specific primers were designed based on the differences between the sheep mitochondrial cytochrome b gene (CYTB) and the duck cytochrome c oxidase subunit III (COX3). The dRT-PCR experiment was conducted using the rapidly extracted animal tissue DNA. By analyzing the melting curve, the sheep and duck-derived components were qualitatively identified according to the differences in peak temperature. Results The optimized method for crude extraction of animal tissue DNA could extract 586 ng/μL of DNA suitable for species detection from 20 mg of animal tissue within 5 minutes. The sheep-derived gene products and duck-derived gene products produce melting peaks at 84.79 ℃ and 81.66 ℃ respectively, with no specific amplification for non-target species. It could quickly detect more than 0.01 ng/μL of crude duck DNA within 1 hour. Conclusion This method is simple to operate, low in cost, and has high sensitivity, providing effective technical support for the identification of adulteration in mutton products.