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Food safety is an important point in consuming food, in order to produce good health for the community. But lately there were many foods circulating in the community that are no longer guaranteed to be safe, namely the existence of counterfeiting and mixing beef with other meats such as rat meat. Rat meat is forbidden for Muslims to consume. In addition, rat can be a reservoir for several disease-causing pathogens in humans. This makes the majority of the Muslim community in Indonesia very worried and in dire need of guarantees for safe and halal food. The purpose of this study was to develop an in-house method for detection of rat meat-contaminated beef-based food product using the multiplex PCR method. Multiplex PCR in this study was optimized on the three variation of PCR program. The optimum multiplex PCR conditions to amplify the ND5 gene of rat and beef simultaneously was using the combination of cycling program of Mur-MA primer pair and Bos primer pair that resulting 919 bp and 206 bp DNA bands which were amplicons of therat and bovine ND5 gene, respectively.

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