一对同时鉴定8种动物源性成分的通用引物的制备及应用

肉类真假鉴定是食品检测工作的内容之一，目前已有多种基于PCR的肉类鉴定方法，但是鉴定种类和效率受限。本研究设计了一对基于普通PCR技术可同时鉴定8种动物源性成分的通用引物并建立了鉴定方法。该引物以线粒体DNA为靶标，利用扩增产物中不同物种间的插入缺失多态性片段大小即可鉴定山羊、绵羊、鹿、水牛、牛、牦牛、猪和骆驼8个物种，扩增后分别得到728 bp、704 bp、504 bp、453 bp、448 bp、431 bp、396 bp和326 bp的片段，每种PCR产物经SspI酶切后产生数量和大小不同的片段，可以进一步清晰鉴别8个物种。引物特异性测试表明和其他常见肉类动物DNA无交叉反应，DNA检测最低限度在0.01~0.05 ng。应用本方法对40份市场肉类及产品的检测表明，羊肉串、羊肉卷以及特色畜产品如驼肉、鹿肉和驴肉存在较多的掺假行为。与其他现有PCR检测方法相比，该方法具有简便易行和高通量的优点，可以作为肉类掺假筛选检测的常规方法。

Preparation and Application of a Pair of Universal Primers for the Simultaneous Detection of Eight Animal-derived Ingredients

Identification of fraud in meat products is one of the aims of food inspection. Many meat identification methods have been developed based on polymerase chain reaction (PCR), but the number of species and detection efficiency are limited. In the present study, a pair of universal primers was designed for the simultaneous identification of eight animal-derived ingredients and a corresponding detection method was developed. Mitochondrial DNA was used as the target of this pair of primers, and insertion-deletion polymorphisms (variation in the fragment length) of the amplified products of different species was used to identify eight species, including goat, sheep, deer, buffalo, cattle, yak, pig, and camel. The PCR amplifications generated 728 bp, 704 bp, 504 bp, 453 bp, 448 bp, 431 bp, 396 bp, and 326 bp length fragments from goat, sheep, deer, buffalo, cattle, yak, pig, and camel in a single amplification reaction, respectively. The eight PCR products could be cut into different numbers of fragments with different lengths using the restriction enzyme SspI, to further distinguish between the eight species. The primer specificity test indicated that this assay had no cross-reactivity with other common meat animals. The detection limit of the DNA samples for eight animal species varied from 0.01 to 0.05 ng. The developed assay was applied to the analysis of 40 commercially available meat products, and the results showed that adulteration often occurred in mutton rolls, mutton kebabs, and other characteristic animal meats, such as deer meat, camel meat, and donkey meat. Compared with other existing PCR-based technologies, this method is a simple high throughput assay that can be used in routine screening for the detection of fraud and adulteration in various meat food products.