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Journal of Food Technology
Year: 2006  |  Volume: 4  |  Issue: 2  |  Page No.: 160 - 165

Development and Evaluation of Nested PCR for Specific Identification of Swine-Derived Products in Processed Food and in Animal Feed Concentrates

Khairalla M.S. Khairalla, Imadeldin E. Aradaib, Tigani Hassan, Ali A. Majid, Abdelrahim E. Karrar, Ahmed M. A. Osman and Osman A. Osman    

Abstract: A nested Polymerase Chain Reaction (PCR) assay for specific identification of pork or swine-derived products in processed food and in animal feed concentrates was developed and evaluated. The mitochondrial cytochrome-b (mtcyt-b) gene was used as a target DNA for PCR amplification. Two pairs of primers (PSL1 and PSR2) and (PSL3 and PSR4), were used for the nested PCR in two amplification steps. First the outer pair of primers (PSL1 and PSR2), derived from a highly conserved region of swine mtcyt-b gene, produced a 1055 base pair (bp) PCR amplicon from swine DNA. Amplification products were visualized on ethidium bromide-stained agarose gels from 100 fg of swine DNA equivalent to 1000 copies of mtcyt-b gene. The nested primers (PSL3 and PSR4) produced a 361 bp PCR product, internal to the annealing sites of primers (PSL1 and RSR2). The nested amplification confirmed the identity of the primary amplified PCR product and increased the sensitivity of the PCR assay. The nested PCR with ethidium bromide-stained agarose gels detected the amount of as little as 0.001 fg of DNA (equivalent to a single copy of Swine-mtcyt-b gene). The specificity studies indicated that neither the primary 1055 bp nor the nested 361 bp PCR products were detected from DNA extracted from a variety of other animal species including, sheep, goat, cattle, deer, camel, horse, donkey, chicken and fish. Application of this nested PCR to processed food including, fresh pork, smoked ham, marinated pork, canned luncheon, petís food, poultry feed resulted in amplification of the swine specific PCR products. The described nested PCR provides a valuable tool to authenticate the presence of swine-derived product in processed food and in commercial animal feed concentrates.

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