Published in Scientific Bulletin. Series F. Biotechnologies, Vol. XXI
Written by Anat DENYINGYHOT, Chirapiphat PHRAEPHAISARN, Mongkol VESARATCHAVEST, Winai DAHLAN, Suwimon KEERATIPIBUL

Halal food approved for Muslim consumers is strictly noticed that avoiding from contaminations that do contradict from Islamic regulations, such as the contamination of animal forbidden by Halal standards. Importantly, High resolution melting analysis (HRMA) is a potential molecular technique, which is used for identifying the species of organisms. Therefore, the research objective is to use the HRMA technique for simultaneous detection of the three forbidden animals (pigs, dogs, and rats) that have high opportunity to be adulterated in Halal food. The HRMA, targeting a fragment of NADH dehydrogenase (ND5), ATPase subunit 6 (ATP6), and Cytochrome B (Cytb) genes, were developed in order to authenticate the forbidden animal and their mixtures. Species-specific primers were designed and combined in a multiplex HRMA resulting in different sequences and therefore different melting behaviours for each species. The multiplex HRMA was then evaluated the PCR specificity against the targeted DNAs of targeted and nontargeted. It’s demonstrated that the method had no cross-reaction with DNA from the experimental animal species (pigs, dogs, rats, cats, monkeys, chickens, horses, sheep, goats, and donkeys). The HRMA profile of amplified amplicons from the targeted animals produced uniquely melting peaks that were easily distinguished for each species in this study. Taken together, all data indicates that this multiplex HRMA is a simple, fast, specific, and cost-effective detection method for pig, dog, and rat in halal food. In order to carry out the analysis of commercial food products, 150 commercial food products was used to screen by species-specific primers for pigs, dogs, and rat were combined in multiplex HRMA. This method revealed that one sample was contaminated with pigs’ DNA in the examined products. Therefore, the HRMA could be used as a halal verification technique for detecting aforementioned forbidden animals contaminated in halal food products.

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