Development and Evaluation of Pcr Assays for Detection of Animal-Derived Products in Processed Food and Animal Feed Concentrates
Development and Evaluation of Pcr Assays for Detection of Animal-Derived Products in Processed Food and Animal Feed Concentrates
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Date
2015-04-15
Authors
Mohamed, Khairalla
Journal Title
Journal ISSN
Volume Title
Publisher
University of Khartoum
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.
Description
103 Pages
Keywords
Pcr Assays, Detection ,Animal,Derived Products,Processed,Food,Animal,Feed,Concentrate