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目的:建立马、牛、猪、鸭肉四重聚合酶链式反应(PCR)反应体系,为牛肉掺假检测提供参考。方法:选取马、牛、猪、鸭肉阳性样本,根据线粒体基因组中细胞色素b(cytochrome b)序列,设计多重PCR引物,同时对马、牛、猪、鸭肉进行PCR及电泳检测,建立四重PCR反应体系,并运用该体系对市场40份牛肉制品进行检测。结果:引物比例为马:牛:猪:鸭=1:1:2:1时,46℃退火温度下反应35个循环,四重PCR效果较好;建立四重PCR反应体系,四重PCR条带清晰且亮度较为一致,几乎没有二聚体产生,扩增灵敏度高。根据样品检测结果,烤牛肉串、牛肉丸的掺假率较高,分别为36.4%、75%,牛排未检出除牛肉外的三种动物源成分,但不排除有其他动物源成分的掺入。结论:四重PCR反应体系可用于牛肉掺假检测。
Objective: To establish a quadruple polymerase chain reaction (PCR) reaction system for horse, cow, pig and duck to provide a reference for the detection of beef adulteration. Methods: The positive samples of horses, cattle, pigs and ducks were selected. According to the sequence of cytochrome b in mitochondrial genome, multiple PCR primers were designed. The PCR, PCR reaction system, and the use of the system on the market 40 beef products for testing. The results showed that the quadruple PCR was the best when the ratio of primers was horse: cow: pig: duck = 1: 1: 2: 1, and the reaction was performed at 46 ℃ for 35 cycles. With a clear and consistent brightness, almost no dimer, amplification sensitivity. According to the test results of the samples, grilled beef skewers and beef balls had higher adulteration rates of 36.4% and 75%, respectively, while the steak did not detect three animal source components other than beef but did not exclude other animal source components Into. Conclusion: Quadruple PCR reaction system can be used to detect beef adulteration.