By: Rahayu WP1,2, Panggabean RIL1, Nikastri E1, Pusparini N1
1Research Center for Drug and Food, National Agency for Drug and Food Control,
2Department of Food Science and Technology-Bogor Agricultural University
Development in food safety analysis especially for detection a pathogen is challenging in food safety assessment system. DNA identification based on PCR (Polymerase Chain Reaction) is one of the identification methods with its high accuracy and precision. The aim of this research was to develop PCR procedure for identifying DNA of pathogenic bacteria isolated from ready to eat food product. S. aureus and E. coli were chosen as bacteria model. Isolation and identification of bacterial DNA from food’s matrices (protein, carbohydrate, and fat) and ready to eat food were carried out. Modified illustrate Bacteria Prep Mini Spin’s Kit for DNA isolation and amplification was used as a tool in this research. To extract the DNA Cethyl Trimethyl Ammonium Bromide (CTAB) and Phosphate Buffer Saline (PBS) were used. Universal primer (Eco-1, F:5’-gac ctc cgt tta gtt cac aga -3’ and Eco-2, R:5’-cac acg ctg acg ctg acc a-3’) were used for PCR reaction. DNA amplification started with pre-denaturation (2’, 95OC), 35 cycles denaturation step (2’, 95OC), annealing step (1’, 54OC), extension step (1’, 72OC) and final extension (5’, 72OC). PCR results were analyzed by gel agarose 1.5 % and RNAas free water was used for negative control to avoid a positive false. The results indicated that DNA of S. aureus and E.coli could be isolated and identified with this modified procedure. The differences from previous methods were the addition of lysozyme to break cell wall of positive Gram bacteria. However, the variety of food’s matrices showed a different result of DNA bands. More complex of food’s matrices resulted in more difficulties in the isolation of DNA bacteria from food.
Keywords: S. aureus, E. coli, food’s matrices, primer, PCR
Presented at 11th ASEAN Food Conference, Brunai Darusalam, October 21-23th2009