动物产品中猪源性和牛源性成分双重荧光PCR检测方法的建立

Identification of Swine-derived and Bovine-derived Materials in Animal Products by Multiplex Fluorescent PCR Assay

[目的]建立双重荧光PCR法,检测动物产品中猪、牛源性成分。[方法]分别针对猪、牛线粒体DNA(mitochondrial DNA,mtDNA)种间保守基因,设计特异性引物与探针,通过对反应体系和反应条件的优化筛选,建立了双重荧光PCR方法,在同一个荧光PCR反应中完成2种动物源性成分的检测,并对该方法的特异性、敏感性进行评估。[结果]对16种不同的动物DNA进行检测,仅猪、牛源性成分收集到相应的典型的S型扩增曲线,对猪、牛二联模板的检测,可同时收集到相应模板的扩增曲线,其余14种动物源性成分未发现扩增曲线。双重荧光PCR对猪肉、牛肉模板的最低检出限均为10-5,与相应的单重荧光PCR方法的检出限一致。[结论]该方法特异性强,敏感性高,适用于肉制品、奶制品、饲料等动物源性产品的检测。

[Objective ] To develop a multiplex fluorescent PCR assay method to detect swine-derived and bovine-derived materials in animal products. [ Method ] Two pairs of specific primers and two fluorescent-labled probes were designed and synthesized according to mitochondrial DNA gene sequences. The multiplex fluorescent PCR reaction system was established through the optimized screening of reaction system and reaction conditions, and swine-derived and bovine-derived materials in animal products were detected in the same fluorescent PCR reaction system. Finally, the sensitivity and specificity of the assay were evaluated. [ Result ] The method successfully detected swine-derived and bovine-derived materials from 16 animals. Swine-derived and bovine-derived materials could obtain responsible S-type amplification curve, while there was no amplification curve from the other 14 animals. The detection limits of the assay for swine and bovine were either up to 10^-5, which was consistent with that of single-weight fluorescent PCR assay method. [Conclusion ] The multiplex fluorescent PCR developed in the study has strong specificity and good sensitivity, so it is suitable for the detection of swine-derived and bovine-derived materials in feed, meat products, milk products, etc..