依据牛种属的线粒体细胞色素b(cytochrome b,Cytb)基因上的保守序列设计牛源特异性引物对,建立一种特异性强、灵敏性高、耗时短的鉴定食品中牛源性成分的聚合酶链式反应(polymerase chain reaction,PCR)方法。选取牦牛、水牛、黄牛品种及羊、猪、鸡、鸭、鹅、兔、马、驴、鱼、大豆、玉米等动植物源成分进行特异性试验。结果显示:所建立的方法高度特异于牛源性检测,其他非牛动植物源成分均无扩增条带。采用常见的牛品种进行灵敏性试验,结果显示所建立的方法可检测到的最低牛DNA量为0.2 pg;将牛肉分别与猪肉、驴肉、羊肉、大豆组织混合进行PCR测定,结果表明最低检测限为鲜样品含0.01%质量分数的牛源成分,高压121 ℃、0.1 MPa,20 min处理样品含0.01%质量分数的牛源成分。对市售食品中牛源性成分检测结果与现行PCR牛源性成分检测标准方法的检测结果100%一致。
To establish a highly specific, sensitive, and time-saving polymerase chain reaction (PCR) method to identify bovine-derived ingredients, a set of primers were designed, based on conserved sequence of the mitochondrial cytochrome b gene (Cytb) of bovine species. The specificity of the method was evaluated by analyzing three kinds of cattle (yak, buffalo, scalper), and several other common tissues (mammalians, ruminants, avian, vegetation). The results showed that the method was highly specific for bovine-derived ingredients, as non-bovine ingredients were not amplified. The sensitivity of the method was evaluated by using common cattle breeds. The results showed that the minimum amount of bovine DNA that could be detected was 0.2 pg (fresh meat). The beef was mixed with pork, donkey, lamb, and soybean tissues, respectively, for PCR analysis. The results indicated that the lowest detection limits of bovine-derived ingredients in both fresh samples and also in samples that were autoclaved at 121 ℃ under 0.1 Mpa for 20 min were 0.01% mass fraction. The test results of commercial foods tested by this method were completely consistent with current standard methods for detecting bovine-derived ingredients.
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