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

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...

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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
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spelling	my-ums-ep.71372024-05-10T02:51:39Z Development of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) based techniques for meat identification 2008 Lim, Chelven Ai Chen TX341-641 Nutrition. Foods and food supply 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 TOPOÂ® 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. 2008 Thesis https://eprints.ums.edu.my/id/eprint/7137/ https://eprints.ums.edu.my/id/eprint/7137/1/24%20PAGES.pdf text en public https://eprints.ums.edu.my/id/eprint/7137/2/FULLTEXT.pdf text en validuser masters Universiti Malaysia Sabah Biotechnology Research Institute (BRI)
institution	Universiti Malaysia Sabah
collection	UMS Institutional Repository
language	English English
topic	TX341-641 Nutrition Foods and food supply
spellingShingle	TX341-641 Nutrition Foods and food supply Lim, Chelven Ai Chen Development of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) based techniques for meat identification
description	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 TOPOÂ® 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.
format	Thesis
qualification_level	Master's degree
author	Lim, Chelven Ai Chen
author_facet	Lim, Chelven Ai Chen
author_sort	Lim, Chelven Ai Chen
title	Development of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) based techniques for meat identification
title_short	Development of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) based techniques for meat identification
title_full	Development of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) based techniques for meat identification
title_fullStr	Development of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) based techniques for meat identification
title_full_unstemmed	Development of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) based techniques for meat identification
title_sort	development of polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) based techniques for meat identification
granting_institution	Universiti Malaysia Sabah
granting_department	Biotechnology Research Institute (BRI)
publishDate	2008
url	https://eprints.ums.edu.my/id/eprint/7137/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/7137/2/FULLTEXT.pdf
_version_	1804890333601857536

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

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