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