12种动物源性成分检测方法的建立及标准样品研制

Establishment of detection methods for 12 kinds of animal-derived ingredients and development of reference materials

中文关键词:  肉及肉制品  动物源性成分  实时荧光定量聚合酶链式反应  标准样品  方法建立

英文关键词:meat and meat products  animal-derived ingredients  quantitative real-time polymerase chain reaction  reference material

中文摘要:

      目的 建立一种可同时检测食品中猪、黄牛、水牛、牦牛、山羊、绵羊、鸡、鸭、鹅、驴、马、兔12种动物源性成分的高通量实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qPCR)方法, 并研制相应的标准样品。方法 以各动物源性成分的特异性靶基因构建引物和探针及反应体系, 评价其灵敏度、特异性, 同时研制相应的标准样品。结果 本研究构建的一套可同时检测12种动物源性成分的qPCR方法; 灵敏度均可达0.01 ng/μL, 除相应源性成分外其他物种无扩增现象, 特异性良好; 该方法对生鲜肉中的不同源性成分的检出限为0.1%。配套研制的标准样品, 均匀性和稳定性良好, 特性值为相应动物源性成分阳性, –18 ℃条件下冷冻有效期可达36个月。结论 本研究建立的方法具有特异性好、灵敏度高, 可有效提升检测效率, 降低检测成本; 制备的标准样品能够满足标准样品的要求, 可用于检测全程的质量保证等预期用途。

英文摘要:

      Objective To establish a method for the simultaneous detection of 12 kinds of animal-derived ingredients in food, including Sus scrofa, Bos taurus, Bubalus bubalis, Bos grunniens, Capra hircus, Ovis aries, Gallus gallus, Anas platyrhynchos, Anser cygnoides, Equus asinus, Equus caballus, Oryctolagus cuniculus by high-throughput quantitative real-time polymerase chain reaction (qPCR), and develop corresponding reference materials. Methods Primers, probes and reaction system were designed based on specific target genes of each animal-derived ingredient. The sensitivity and specificity of the method were evaluated. Concurrently, corresponding reference materials were developed. Results A qPCR system for the simultaneous detection of 12 kinds of animal-derived ingredients was successfully established. The sensitivity for each ingredient was 0.01 ng/μL, and no amplification was observed in non-target species, demonstrating good specificity. The limit of detection of this method for different animal-derived components in fresh meat was 0.1%. The developed reference materials exhibited good homogeneity and stability, with the assigned value being positive for the corresponding animal-derived ingredients. The shelf life under frozen conditions at –18 °C is up to 36 months. Conclusion The established method exhibits good specificity and high sensitivity, effectively improving detection efficiency and reducing costs. The prepared reference materials meet the requirements for reference materials and can be used for intended purposes such as quality assurance throughout the detection process.