Cloning of Pork NADH Dehydrogenase Subunit 4 (ND4) Gene as Internal Positive Control Candidate for Food Halal Authentication based on PCR Method
J. Kusnadi(1*), E L Arumingtyas (2), K U Al Awwali (3), and A S Zakiyah (2)

1Department of Food Science, Faculty of Agricultural Technology, Universitas Brawijaya, Indonesia
2Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya, Indonesia
3Department of Animal Product Technology, Faculty of Animal Science, Universitas Brawijaya, Indonesia

Abstract

In recent years, Muslim consumers very concerned about the adulteration cases in halal meat with non-halal meat on food product. These cases make the halal food identification process important for Muslim community. The effective detection methods to identify non-halal meat contamination in food is by PCR or qPCR. The possibility of the emergence of false positives and negatives in identifying non-halal DNA contamination in food by PCR causes the need for Internal Positive Control (IPC). IPC acts as a detector for errors in the PCR process. This research aims to develop IPC using cloning technique for pork contamination detection in food based on PCR method. The whole DNA were isolated from pork belly meat. The DNA then amplified using specific primer of ND4 gene for Pork species using PCR. The ND4 gene was ligated into the pGEM-T cloning vector and transformed into E. coli JM109 cells. The characterization process of the cloned results by recombinant plasmid isolation and PCR. We successfully inserted the ND4 gene into the pGEM-T cloning vector, indicated by the growth of 11 white colonies of bacteria on culture plate. The characterization process result showed the successfully inserted DNA was the ND4 gene with 120 bp length.

Keywords: Cloning, Halal, ND4 gene, Pork

Topic: Food safety and security