多重PCR技术在食品中4种肉类成分快速鉴别中的应用

Application of multiplex multiplex PCR technology in rapidly identifying 4 kinds of meat

目的:探究多重PCR技术在食品中4种(猪肉、牛肉、羊肉和鸡肉)肉类成分快速鉴别中的应用。方法:采用DNeasy试剂盒提取法、SDS-蛋白酶K法与CTAB-蛋白酶K法提取肉类中总DNA,比较三种方法的效率及纯度,确定最优方式。在动物线粒体细胞色素b基因的差异性位点,设计两套多重PCR引物。建立并优化多重PCR反应体系,根据电泳检测扩增产物分子量差异以快速鉴别4种肉类。结果:DNeasy试剂盒法、SDS-蛋白酶K法提取DNA效率显著优于CTAB-蛋白酶K法,P<0.05。在优化反应条件下选择特异性高、序列长的多重PCR引物可有效快速鉴定食品中猪、牛、羊与鸡源性成分,检测灵敏度为皮克级。结论:多重PCR方法应用于食物中多动物源性成分鉴别,精确稳定快速。

Objective： To explore the rapid identiifcation for 4 kinds of meat （pork, beef, mutton and chicken） in food by multiplex PCR technique. Methods： The total DNA was extracted by DNeasy kit extraction, SDS-protease K and CTAB-protease K; the effciency and purity of the 3 methods were compared; then the optimal method was determined. 2 sets of multiplex PCR primers were designed in the mitochondrial cytochrome b gene of animals. The multiplex PCR reaction system were established and optimized, to rapidly identify 4 kinds of meat according to the difference of molecular weight by electrophoresis. Results： The extraction effciency of DNeasy kit method, SDS-protease K is significantly better than the CTAB-protease K method, with statistical difference,P〈0.05. On the optimal reaction conditions that high speciifcity, sequence length of the multiplex PCR primers can be effectively and rapidly identify food ingredients of pigs, cattle, sheep and chicken; the detection sensitivity is peake. Conclusion： Application of multiplex PCR technology in identifying meat is accurate, stable and quick.