DEVELOPMENT OF TECHNIQUES BASED ON POLYMERASE CHAIN REACTION- RESTRICTION FRAGMENT LENGTH POLYMORPHISM (PCR-RFLP) FOR MEAT IDENTIFICATION (HALAL & NON-HALAL)

A molecular analysis based on Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene for meat identification has been developed. The genomic DNA of known identities of meats were extracted and subjected to PCR amplification targeting the mt cyt b gene. The analysis produced PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs. Species identification was determined by digesting the amplicons with different types of restriction endonucleases which generates species-specific eletrophoresis banding patterns. The PCR-RFLP profile of all the species tested provide standards and significant data for interand intraspecies identification for this study. The threshold of detection of the PCR-RFLP method was carried out using blended pork and chicken meats which were mixed in different percentages, 0.1%, 0.2% 0.3%, 0.4%, 0.5%, 1%, 5%, 10%, 20%, and 100% respectively. The results indicated that the PCR-RFLP method was sensitive enough to detect down to 0.1% of contaminant. Sequencing the PCR products was performed to confirm the cyt b gene of various meats. INTRODUCTION The Government intends to make Malaysia one of the major halal food distributors in the world. Thus, issues about food authenticity and adulteration have been a major concern. In the production of halal meat, raw and processed meats is separated from non-halal meat such as pork. Nevertheless, contaminations from other meats sometimes occur unintentionally or accidentally. Therefore, a technology that can identify the meat identity in food is required. Detection technique that is developed is also advantageous to followers of other religions and clinical patients with strict nutritional diets. Several meat identification techniques based on protein from meats have been developed. But, these techniques have some disadvantages namely; they are not very sensitive and are unable to detect severely heat-treated meat due to protein denaturation during processing. A molecular technique based on DNA is more sensitive and accurate because DNA is a remarkably stable molecule allowing analysis of processed and heat-treated food products (Unseld et al., 1995). The information content of DNA is also much greater than protein (Wolf et al., 2000). Cytochrome b (cyt b) gene is found in the mitochondrial genome of all meats and it has been shown to be polymorphic among different animals. The cyt b gene can be isolated by PCR amplification using a set of universal PCR primers, CYTb1 and CYTb2, which are capable of amplifying cyt b gene producing a fragment with the size of approximately 360 bp from all types of meats. The amplicons are digested with restriction endonucleases to produce DNA fragments of different sizes and banding pattern on the agarose gel for the study of interspecies polymorphisms. PCR amplifies target region of template DNA in a rapid and sensitive manner (Saiki et al., 1988). PCR-RFLP has shown to be a technique for identification of meat product. This method had been tested and successfully identified raw meat (Bellagamba et al., 2001). Therefore, the aims of this project are to develop a molecular analysis method based on Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) cyt b