title: Double Gene Targeting Multiplex Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Assay Discriminates Beef, Buffalo, and Pork Substitution in Frankfurter Products.
abstract: Beef, 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.
authors: M A Motalib
authors: MA
authors: Hossain
authors: Md Eaqub
authors: ME
authors: Ali
authors: Sharifah Bee
authors: SB
authors: Abd Hamid
authors: Asing
authors: Institute of Halal Products Research, Universiti Putra Malaysia , 43400 UPM Serdang, Selangor, Malaysia.
authors: Shuhaimi
authors: S
authors: Mustafa
authors: Institute of Halal Products Research, Universiti Putra Malaysia , 43400 UPM Serdang, Selangor, Malaysia.
authors: Mohd Nasir
authors: MN
authors: Mohd Desa
authors: Deparment of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University , Kuantan 25200, Pahang, Malaysia.
authors: I S M
authors: IS
authors: Zaidul
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis / Q000379:methods*; D000818:Animals; D002020:Buffaloes / Q000235:genetics*; D002417:Cattle / Q000235:genetics*; D016002:Discriminant Analysis; D005506:Food Contamination / Q000032:analysis*; D008461:Meat Products / Q000032:analysis*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D013552:Swine / Q000235:genetics*
keywords: double gene-targets; molecular fingerprints; multiplex PCR-RFLP; species-specific identification