Development of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) based techniques for meat identification

• Main Author: Lim, Chelven Ai Chen
• Format: Thesis
• Language: English; English
• Published: 2008
• Subjects: TX341-641 Nutrition. Foods and food supply
• Online Access: https://eprints.ums.edu.my/id/eprint/7137/1/24%20PAGES.pdf; https://eprints.ums.edu.my/id/eprint/7137/2/FULLTEXT.pdf; https://eprints.ums.edu.my/id/eprint/7137/

The primary objective of the research was to develop technique to identify various types of meats and to determine the detection of threshold levels of the technique. This technique is 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 and authentication. Firstly, meat samples of pork, lamb, ostrich, cattle, buffalo, chicken and turkey were sampled randomly from commercial establishment in Sabah. The genomic DNA of known identities of meats were extracted and were subjected to PCR amplification by using the Specific primers CYTb1 and CYTb2 targeting the mt cyt b gene. PCR amplification generated an amplicon with approximate size of 360 bp. The amplicon was doned onto a TOPO® TA 2.1 plasmid, and sequenced. Sequences derived from these meats were aligned using SDSC biology workbench to determine homologous regions. The results of the alignment confirmed the identities of the meat species, and these sequences were used as references for the subsequent meat analyses and for future meat identification. Secondly, meat species identification was conducted by digesting the amplicons with a range of restriction endonudeases namely, AluI, BsaJI, BstNI, BstUI, NsIi, RsaI, TaqI, which generates species-specific eletrophoresis banding Patterns. The PCR-RFLP profile of all the species tested were used to construct a reference library and to provide a significant data for inter- and intra species identification for this study. Thirdly, the threshold of detection using the PCR-RFLP method was tested using blended pork and chicken, or pork and beef which were mixed In different percentages, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 5%, 10%, 20%, and 100% respectively. Results indicated that the PCR-RFLP method was sensitive enough to detect down to 0.1% of meat contaminant. Finally, processed meat products procured from various supermarkets were analyzed and authenticated using the technique developed and PCR conditions optimized in this study. The results of the analyses indicated that some of the meat labels did not reflect the content as claimed by the manufacturer. For instance, one of the processed meat products tested which was labeled as beef product was found to be contaminated by chicken meat while the raw beef were dearly not as it is claimed.