同步检测动物源性成分的四重PCR的条件优化和检出限分析

Optimization of quadruple PCR for simultaneous detection of animal-derived ingredients and its detection limit analysis

为建立肉制品中牛、羊、猪、鸡动物源性成分的快速高效多重PCR检测方法,利用4对牛、羊、猪和鸡源特异性PCR引物(靶基因序列来源于线粒体基因组)对多重PCR反应条件进行优化,并确定该检测方法的检出限。结果表明:四重PCR方法最佳反应条件为牛、羊、猪、鸡源特异性引物浓度分别为0.16μmol∕L、0.40μmol∕L、0.16μmol∕L、0.16μmol∕L,退火温度58℃,退火时间45 s,循环次数40。在最佳反应条件下,所建立的牛、羊、猪和鸡4种动物源性成分四重PCR的g DNA检出限为25 pg。应用优化后的四重PCR方法对20份市售肉制品样本进行盲样检测,验证鉴别方法的准确性,结果与现行标准的检测结果一致,掺假率达45%。本试验为该4种动物源性成分的快速检测提供了技术支撑。

In order to establish a rapid and efficient multiple PCR method for animal derived ingredientssuch as beef,mutton,pork and chicken,the specific PCR primers of cattle,sheep,pig and chicken,which werederived from the mitochondrial genome, were used to optimize the multiple PCR reaction condition, and thedetection limit of the method was determined. The results showed that the optimum reaction condition of themultiple PCR method were 0. 16 μmol∕L,0. 40 μmol∕L,0. 16 μmol∕L,0. 16 μmol∕L for primers of cattle,sheep,pig and chicken derived ingredients respectively,the annealing temperature was 58 ℃,the annealing time was45 s,and the number of cycles was 40. Under the optimal reaction condition,the detection limit of g DNA for the4 animal derived ingredients was 25 pg in quadruple PCR. Using the optimized quadruple PCR method,20 samples of marketed meat products were detected by blind sample,and the accuracy of the identification methodwas verified. The results were consistent with the test results of the current standard,and the adulteration rate was45%. This study provided technical support for rapid detection of the 4 animal derived ingredients.