食品中4种肉类成分多重PCR的快速鉴别方法

doi:10.3864/j.issn.0578-1752.2012.09.024

摘要/Abstract

摘要： 【目的】建立快速可靠的用于食品中4种肉类（猪肉、牛肉、羊肉和鸡肉）成分快速鉴别的通用引物多重PCR（universal primers-multiplex PCR，UP-M-PCR）方法。【方法】使用DNeasy试剂盒提取法、SDS-蛋白酶K法和CTAB-蛋白酶K法分别提取肉类中总DNA，通过比较提取效率和纯度，确定提取肉类中总DNA的方法；基于动物线粒体细胞色素b基因的差异性位点，设计两组各5条长度不同的多重PCR引物，建立并优化多重PCR反应体系，通过电泳检测扩增产物分子量差异实现4种肉类的快速鉴别；应用优化的多重PCR方法对80份市售食品样本进行盲样检测，验证鉴别方法的准确性。【结果】SDS-蛋白酶K法与DNeasy试剂盒提取法的DNA效率显著优于CTAB-蛋白酶K法；在优化的反应条件下，选择特异性高、序列较长的多重PCR引物可有效地进行食品中猪、牛、羊和鸡源性成分的快速鉴定，检测灵敏度达到皮克级DNA；对市售食品样本的鉴别验证了方法的实用价值。【结论】多重PCR方法精确稳定，可用于食品中多种动物源性成分的快速鉴别。

关键词: 多重PCR, 鉴别, 猪肉, 牛肉, 羊肉, 鸡肉

Abstract: 【Objective】An accurate and reliable universal primers-multiplex PCR (UP-M-PCR) method was developed to identify 4 kinds of meat ingredient (chicken, beef, lamb, pork) in food products.【Method】 Total DNA in meat samples were extracted using a commercial DNA extraction kit, a CTAB-Proteinase k method and a SDS-Proteinase k method, respectively. The concentration and purity of DNA were evaluated to select the optimal method. Two sets of UP-M-PCR primers in different lengths were designed based on the homologous and specific sites of mitochondrial cytochrome b gene. Each set was comprised of 5 primers: one general forward primer and four species-specific reverse primers. The UP-M-PCR system, followed by gel electrophoresis assay of speces-specific bands, was established for meat identification. The meat spices were identified according to different lengths of PCR products. Meanwhile, the accuracy of the method was evaluated by testing of eighty food samples from the market. 【Result】The SDS-Proteinase K method and the DNA commercial kit presented better extraction efficiencies in contrast to the CTAB-Proteinase K method. The optimized UP-M-PCR was established using the set of longer primes, which showed higher specificity than that using the shorter ones. The detection limit was picograms of DNA. The accuracy of the method was verified by examination of 80 food samples from the market. 【Conclusion】A sensitive, reliable and convenient method was developed to identify 4 kinds of meat ingredient in food products.

Key words: multiplex PCR, identification, pork, beef, lamb, chicken

何玮玲, 张驰, 杨静, 黄明, 杨军. 食品中4种肉类成分多重PCR的快速鉴别方法[J]. 中国农业科学, 2012, 45(9): 1873-1880.

HE Wei-Ling, ZHANG Chi, YANG Jing, HUANG Ming, YANG Jun. A Quick Multiplex PCR Method for the Identification of Four Meat Ingredients in Food Products[J]. Scientia Agricultura Sinica, 2012, 45(9): 1873-1880.

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