Development and Evaluation of Pcr Assays for Detection of Animal-Derived Products in Processed Food and Animal Feed Concentrates

Abstract

Nested (nPCR) and semi-nested polymerase chain reac tion assays for specific identification of animal-derive d products in processed food and in animal feed concentrates were developed and evaluated. The mitochondrial cytochrome-b (mtc yt-b) gene was used as a target DNA for PCR amplification. Pai rs of primers derived from highly conserved regions of mt cyt-b gene were used for the PCR assays in two amplification s teps. For the specific identification of swine mtcyt-b gene, two pairs of primers (PSL1 and PSR2) and (PSL3 and PSR4), were u sed. The outer pair of primers (PSL1&PSR2) produced a 1055 b ase pair (bp) PCR product from swine DNA. Amplification pro ducts were visualized on ethidium bromide-stained agarose gels from 100 fg of swine DNA equivalent to 1000 copies of mt cyt-b gene. The nested primers (PSL3 and PSR4) produced a 361 b p PCR product, internal to the annealing sites of primers (PSL1 &RSR2). The nested amplification confirmed the identity of the primary amplified PCR product and increased the sen sitivity of the PCR assay. The nested PCR with ethidium bromi de-stained IX 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 prim ary 1055 bp nor the nested 361 bp PCR products were detected fr om DNA extracted from a variety of other animal species in cluding, sheep, goat, cattle, deer, camel, horse, donkey, ch icken and fish. Application of this nested PCR to processed food in cluding, fresh pork, smoked ham, marinated pork, canned lunc heon, pet’s food, poultry feed resulted in amplification of the swine specific PCR products. Semi-nested PCR, a similar technique was also used in this study. It revealed the same results as nPCR when ap plied to cattle mtcyt-b gene using two pairs of primers (RSL 1 and CSR2) and (CSL1 and CSR2) which produced 386bp and 283bp PCR products, respectively.