A simplex and multiplex PCR assay for simultaneous detection of beef, pork, and chicken meat in sausages based on mitochondrial DNA Cytochrome oxidase sub-unit I

Abstract

Adulteration in the processed beef product industry has long been an issue affecting consumers’ rights and legal protection as beef is an expensive raw material. The intense market competition opens up opportunities for manufacturers of processed beef products to counterfeit and mixes their products with other cheap meat ingredients such as chicken and pork. In addition, the use of pork to substitute beef in the production of sausages without proper labelling on the final products is both an adulteration and halal assurance issue in the Muslim-majority market. Multiplex PCR is a sensitive assay used for the identification of raw materials used in the production of processed food and it reduces the cost and time of analyzing a large number of samples. The purpose of this research was to apply the multiplex PCR assay targeting mitochondrial DNA Cytochrome Oxidase Sub-Unit I (COI) sequence and to determine the detection limits for DNA concentrations in beef, pork, and chicken. The DNA genomes that were extracted from each meat were diluted to concentrations of 25; 10; 1; 0.1; 0.01; and 0.001 ng/µL, and then used as a DNA template for the simplex-and multiplex-PCR. The results of simplex-and multiplex-PCR showed that the COI primers used were able to amplify the DNA of bovine, porcine, and chicken accurately as indicated by amplicons of 263 bp, 168 bp, and 596 bp, respectively. It was concluded that the simplex-and multiplex-PCR methods using COI primers can be used to identify bovine, porcine, and chicken DNA isolated from sausages with a sensitivity of 0.001 ng/μL.