Swine-Specific PCR-RFLP Assay Targeting Mitochondrial Cytochrome B Gene for Semiquantitative Detection of Pork in Commer
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Swine-Specific PCR-RFLP Assay Targeting Mitochondrial Cytochrome B Gene for Semiquantitative Detection of Pork in Commercial Meat Products M. Eaqub Ali & U. Hashim & S. Mustafa & Yaakob Bin Che Man
Received: 1 July 2011 / Accepted: 10 August 2011 / Published online: 2 September 2011 # Springer Science+Business Media, LLC 2011
Abstract Verification of pork adulteration in commercial meat products is increasingly important for the authentication of Halal labels in processed foods. Here, we documented a PCR–restriction fragment length polymorphism (RFLP) assay with high precision and reproducibility for the tracing of porcine DNA in commercial meat products. The assay combined the species-specific primers to selectively amplify a short fragment (109 bp) of porcine cytochrome b gene from a heterogeneous background of genomic DNAs followed by RFLP analysis to authenticate real amplicon. The analysis of PCR products and restriction digests was automated in a chipbased capillary electrophoresis incorporated in Agilent 2100 bioanalyzer. The swine specificity of the assay was checked with 11 different meat-providing animal and fish species. Model experiments, mimicking the processed foods, were performed in binary and ternary mixtures after mechanical grinding and prolonged autoclaving. Finally, four types of the most popular finished meat products (meatball, streaky beacon, frankfurter, and burger) which are prevalent in the Malaysian food market were analyzed in order to verify the assay performance. The assay was sensitive enough to detect 0.0001 ng of swine DNA in pure formats and 0.01% (w/w) spiked pork in extensively processed ternary mixture of pork, beef, and wheat flour.
M. E. Ali : U. Hashim Institute of Nano Electronic Engineering (INEE), Universiti Malaysia Perlis, 01000, Kangar, Perlis, Malaysia S. Mustafa : Y. B. Che Man (*) Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia e-mail: [email protected]
Keywords Halal authentication . Ternary mixtures . Chip-based capillary electrophoresis
Introduction Species authentication and food safety are a growing concern in today’s marketplace. As minced meat is added in most of the processed foods (Tanabe et al. 2007), verification of food labeling should ensure food safety (i.e., unexpected occurrence of food allergies) and gain consumer trust, promoting fair trades in local and international markets. Most countries in the world have regulatory bodies to ensure transparency in food labeling (Tanabe et al. 2007; Standard Malaysia 2009; Murugaiah et al. 2009; Saudi Gazette 2011). According to the EC legislation (178/ 2002) on food safety (European Commission 2002), manufacturers are obliged to clearly label all raw materials used in the preparation of food products. Inspired by the ever-increasing demands for transparency in food industry, a myriad of reports on methodology development for the identification of meat species in processed and raw states have been published in recent years (Tanabe et al. 2007; Murugaiah e
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