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目的建立Taqman实时荧光PCR法对奶制品中掺加物大米、玉米、小麦、高粱、大麦等谷物源性成分的初筛检测体系。方法以常见掺加物大米、玉米、小麦、高粱、大麦等谷物的叶绿体rbcL基因作为靶基因,通过BLAST软件比对,选择其同源保守区域设计引物和探针。经通用性、特异性、灵敏性试验对探针和引物可行性进行验证,并通过模拟含谷物奶粉及市售奶类制品检测对其实际检测能力进行验证。结果所建立体系可扩增大米、玉米、小麦、高粱、大麦等常见的谷物掺加物的DNA提取物,与市售奶制品的主成分牛奶、羊奶以及其常见添加物香蕉、红枣、菠萝、草莓、西红柿、花生、大豆等动植物源性成分无交叉扩增(Ct>35)。对小麦纯DNA提取物检出限为0.01 ng。对分别掺加5种谷物成分的模拟奶制品检出限均可达0.5%,对市售的14份奶类食品中的谷类成分的检测结果均与食品标签相符。结论本研究建立的Taqman实时荧光PCR体系具有通用、相对特异、灵敏、实用等优点,可用于奶制品中掺加或掺杂掺假谷物源性成分的快速定性检测。
Objective To establish a Taqman real-time fluorescence PCR method for screening dairy products, rice, corn, wheat, sorghum, barley and other cereal source components of the screening system. Methods The chloroplast rbcL gene of rice, corn, wheat, sorghum, barley and other cereals was screened by BLAST software. Primers and probes were designed by homologous conservative regions. Proven viability, specificity and sensitivity tests were performed on the feasibility of probes and primers, and the actual detection ability was verified by simulating cereal milk powder and commercial milk products. Results The system was established to amplify DNA extracts of common cereal admixtures such as rice, corn, wheat, sorghum, barley and other cereal extracts, as well as milk, goat milk and their common additives, banana, date, pineapple There was no cross-amplification of animal and plant-derived components such as strawberry, tomato, peanut and soybean (Ct> 35). The detection limit of pure DNA extract of wheat was 0.01 ng. The detection limits of the simulated milk products with 5 kinds of cereal ingredients respectively were up to 0.5%. The test results of the cereal ingredients in 14 milk products on the market were consistent with the food labels. Conclusion The Taqman real-time fluorescent PCR system established in this study has the advantages of generality, relative specificity, sensitivity and practicality. It can be used for the rapid qualitative detection of doping or doping adulterated grains in dairy products.