锁核酸探针技术快速检测鸭源性成分的试验

Taqman-LNA Real Time PCR Assay For Rapid Detecting Duck-Derived Ingredients in Meat and Meat Products

为了建立快速鉴别肉及肉制品中掺假鸭源性成分的改良荧光PCR方法,针对鸭线粒体基因组的保守序列,设计特异性引物和锁核酸(LNA)探针,建立快速鉴别肉及肉制品中掺假鸭源性成分的Taqman-LNA荧光PCR方法,通过设定合理的掺假判定标准,解决肉污染导致的掺假误判。结果建立的方法对鸭肉DNA的检测灵敏度高达1.64 pg;与猪、牛、鸡等11种动物的DNA无非特异性反应;应用该法检测模拟掺假肉制品,结果与预期相符;应用该法检测80份市售肉制品,发现有5份产品掺有鸭源性成分而标签未标示。表明本试验建立的方法特异、敏感,可快速、准确鉴别肉及肉制品中掺假鸭源性成分。

To develop an improved real time PCR assay for rapid detection of duck-derived ingredients in meat and meat products.Species-specific PCR primers and Taqman-LNA probe were designed based on the conserved regions of mitochondrial gene. Taqman-LNA real time PCR assay was developed after optimization of the reaction conditions.Specificity,sensitivity and accuracy of this assaywere tested.The difference between the Ct value of sample and the Ct value of pure duck was considered as a criterion for judgementof meat adulteration to avoid misjudgment of meat polluted by duck meat in processing and selling. And then,this assay was used todetect 80 commercial meat and meat product sample in markets.The detection limit of this assay was 1.64 pg.Specificity of this assaywas tested by analysis of DNA obtained from different animal species and no amplification signal was obtained from DNA of pork,beef,chicken et al.The accuracy of this assay was proved by the test of known added amounts of analyte.We detected duck-derived ingredientsin 5 samples finally and these samples were not marked duck-derived ingredients on the product labels.Conclusion The Taqman-LNAPCR developed is specific and sensitive,and fit for detection of duck-derived ingredients in meat and meat products rapidly andaccurately.