[abstract] COMPARISON OF THREE EXTRACTION METHODS FOR IDENTIFICATION OF Bacillus cereus AND Staphylococcus aureus IN FRIED RICE

By: Yuliangsih S1, Pusparini N1, Khotimah K1, Panggabean RIL1, Rahayu WP1,2

1Research Center for Drug and Food, National Agency for Drug and Food Control, 2Department of Food Science and Technology, Bogor Agricultural University

DNA identification based on Real Time PCR (Polymerase Chain Reaction) is one of rapid methods that can quantify relatively small amount of DNA and monitor the progress of a PCR reaction in real time. In this study, rapid identification of pathogens B. cereus and S. aureus in fried rice was undertaken. DNA isolation of B. cereus and S. aureus was done by comparing three extraction methods: boiling (Medici et al., 2003) and (Hein et al., 2001), phenol chloroform (Sambrook et al., 1989) and commercial Kit (GE Healthcare modified by PROM biotech, 2008). In order to obtain detection limit (LOD) of B. cereus and   S. aureus, both of microbes were diluted in series (10-1-10-8) then artificially inoculated to the sample. DNA Isolate was amplifying by real time PCR using specific primers. Primer BceT1 (5’-GAA TTC CTA AAC TTG CAC CAT CTC G-3’) and BceT2 (5’-CTG CGT AAT CGT GAA TGT AGT CAA T-3’) were used for B. cereus detection (Lindstrom and Mantynen, 1998), while primer SaIF (5′-AAT CTT TGT CGG TAC ACG ATA TTC TTC ACG-3′) and SaIR (5′-CGT AAT GAG ATT TCA GTA GAT AAT ACA ACA-3′) were used for S. aureus detection (Cho et al., 2008). The primer nucleotide sequences were analyzed by Basic Local Alignment Search Tool (BLAST, NCBI).The results indicated that time consumed for DNA extraction of boiling method was faster 3-4 hours than the others, but DNA isolate contained high impurities. Phenol-chloroform method gave more pure DNA isolate but time consumed. Commercial kit method took time longer than the boiling (4-5 hours). Generally, both of boiling and commercial kit methods gave R2 (Intercept) and E (Efficiency) values better and more consistent than phenol-chloroform method. The R2 and E number for S. aureus with boiling method were 0.965 and 78.7% and with commercial kit were 0.920 and 81.2%.  For B. cereus, the R2 and E number for boiling method were 0.944 and 1.3×105% and for commercial kit were 0.894 and 3.6×103%. Detection limit (LOD) of fried rice which was artificially inoculated with B. cereus and S. aureus are 1.3 CFU/ml and 4.4 CFU/ml, respectively.

Keywords: fried rice, identification, real time PCR, Bacillus cereus,Staphylococcus aureus

Presented at Proceeding International Conference on 3th Mathematics and Natural Science (ICMNS), Bandung-Indonesia, November 23-25th 2010

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