jf6b02224_si_006.pdf (158.51 kB)
Double Gene Targeting Multiplex Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Assay Discriminates Beef, Buffalo, and Pork Substitution in Frankfurter Products
journal contribution
posted on 2016-07-18, 00:00 authored by M. A.
Motalib Hossain, Md. Eaqub Ali, Sharifah Bee Abd Hamid, Asing, Shuhaimi Mustafa, Mohd Nasir Mohd
Desa, I. S. M. ZaidulBeef, buffalo, and
pork adulteration in the food chain is an emerging
and sensitive issue. Current molecular techniques to authenticate
these species depend on polymerase chain reaction (PCR) assays involving
long and single targets which break down under natural decomposition
and/or processing treatments. This novel multiplex polymerase chain
reaction–restriction fragment length polymorphism assay targeted
two different gene sites for each of the bovine, buffalo, and porcine
materials. This authentication ensured better security, first through
a complementation approach because it is highly unlikely that both
sites will be missing under compromised states, and second through
molecular fingerprints. Mitochondrial cytochrome b and ND5 genes were targeted, and all targets (73, 90, 106, 120,
138, and 146 bp) were stable under extreme boiling and autoclaving
treatments. Target specificity and authenticity were ensured through
cross-amplification reaction and restriction digestion of PCR products
with AluI, EciI, FatI, and CviKI-1 enzymes. A survey of Malaysian frankfurter
products revealed rampant substitution of beef with buffalo but purity
in porcine materials.