Using Reverse Dot Hybridization Method and 16S rRNA Gene (16S rDNA) for Identifying the Food Poisoning Microorganism in Foods

Reverse dot hybridization 방법과 16S rRNA gene(16S rDNA)을 이용한 식품에서 식중독균의 탐색

  • 김민성 (한양대학교 자연과학대학 생명과학과) ;
  • 신규철 (한양대학교 자연과학대학 생명과학과) ;
  • 이형구 (한양대학교 자연과학대학 생명과학과) ;
  • 한명수 (국가지정 물환경생태복원연구실) ;
  • 민병례 (상명대학교 자연과학대학 생명과학과) ;
  • 최영길 (한양대학교 자연과학대학 생명과학과)
  • Published : 2003.06.01


DNA sequence information on small-subunit rRNA gene (16S rDNA) obtained from food-poisoning bacterial culture was used to investigate the presence of bacterial pathogens in food. By reverse dot blot detection method, presence of food-poisoning bacteria could be confirmed on hybridization of digoxigenin-labeled 16S rDNA Polymerase Chain Reaction (PCR) primer product and biotin-labeled specific oligonucleotide probe. Escherichia coli, Bacillus cereus. and Salmonella sp. were used as the representative food-poisoning bacterial microorganisms. An oligonucleotide probe, based on the variable region of 16S rRNA gene, was used as the specific probe. These tools may be more useful than classic biochemical method for rapid identification of contaminated food.


16S rRNA gene;food-poisoning bacterial;PCR;reverse dot hybridization


  1. Anderson, D.Y., Vredeveld, G.N., Brake, S.R., Buchanan, T.F. and Lewis, J.F. Evaluation of API 20E strips for identification of coagulase negative staphylococci from the urinary tract. Am. J. Med. Technol. 12: 879-881 (1983)
  2. Kawasaki, E., Saiki, R. and Erlich, H. Genetic analysis using polymerase chain reaction-amplified DNA and immobilized oligonucleoide probes reverse dot blot typing. Methods Enzymol. 218: 369-381 (1993)
  3. Sambrook, J. and Russel, D.W. Molecular Cloning: a Laboratory Manual. 3nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, USA (2001)
  4. Reysenbach, A.L., Giver, L.J., Wickham, G.S. and Pace, N.R. Differential amplification for rRNA genes by polymerase chain reaction. Appl. Environ. Microbiol. 58: 3417-3418 (1992)
  5. Stahl, D.A., Flesher, B., Mansfield, H. and Montgomery, L. Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl. Environ. Microbial. 54: 1079-1084 (1988)
  6. Tsen, H.Y., Lin, C.K. and Chi, W.R. Development and use of 16S rRNA gene targeted PCR primer for the identification of escherichia coli cells in water. J. Appl. Microbiol. 85: 554-560 (1998)
  7. Maidak, Cole, B.L., Lilburn, J.R., Parker, T.G., Jr, Saxman, C.T., Stredwick, P.R., J.M., The RDP (Ribosomal Database Project) continues. Nucleic Acids Res. 28: 173-174 (2000)
  8. Muyzer, G., de Waal, E.C. and Uitterlinden, A.G. Profiling of complex microbial populations using denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified gene coding for 16S rRNA Appl.Environ. Microbiol. 59: 695-700 (1993)
  9. Centers for Disease Control and Prevention. Update: multistate outbreak of Escherichia coli O157:H7 infections from hamburgers- western United States, 1992-1993. Morbid. Mortal. Weekly Rep. 42: 258-263 (1993)
  10. Mantero, G., Zonaro, A., Albertini, A., Bertolo, P., Primi, D. DNA enzyme immunoassay: general method for detecting products of polymerase chain reaction. Clin. Chem. 7: 422-429.(1991)
  11. Raskin, L., Stromley, J.M., Rittmann, B.E. and Stahl, D.A. Group-specific 16S rRNA hybridization probes to describe natural communities of methanogens. Appl. Environ. Microbial. 60: 1232-1240 (1994)
  12. Oyarzabal, O.A., Wesley, I.V., Harmon, K.M., Schroeder-Tucker, L., Barbaree, J.M., Lauerman, L.H., Backert, S. and Conner, D.E. Specific identification of Campylobacter fetus by PCR targeting variable regions of the 16S rDNA, Veterinary Microbiol. 58: 61-71 (1997)
  13. Lin, C.K. and Tsen, H.Y. Use of two 16S DNA targeted oligonucleotide as PCR primers for the specific detection of Salmonella in foods. J. Appl. Bacteriol. 80: 659-666 (1996)
  14. Amann, R.I., Ludwig, W. and Schleifer, K.H. Phlogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rew. 59: 143-169 (1995)
  15. ${\O}vre{\aa}s$, L. Forney, L., Daae, F.L. and Torsvik, V Distribution of bacterioplankton in meromictic lake srelcnvannct, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA. Appl. Environ. Microbiol. 63: 3367-3373 (1997)
  16. AIm, E.W., Oerther, D.B., Larsen, N., Stahl, D.A. and Raskin, L. The oligonucleotide probe database. Appl. Environ. Microbiol. 62: 3557-3559 (1996)
  17. Ekins, R., and Chu, F. W. Microarrays; their origins and application.Trends Biotechnol. 17: 217-218(1999)
  18. Bean, N.H. and Griffin, P.M. Foodbome disease outbreaks in the United States, 1973-1987; pathogens, vehicles and trends. J. Food Prot. 53: 804 (1990)
  19. Weisburg, W., Barns, S.M., Pelletier, D.E. and Lane, D.J. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173: 697-703 (1991)
  20. Bavykin, S.G., Akowski, J.P., Zakhariev, V.M., Barsky, V.E., Perov, A.N. and Mirzabekov, A.D. Portable system for microbial sample preparation and oligonucleotide microarray analysis. Appl. Environ. Microbial. 67: 922-928(2001)
  21. Hansen, B.M., Leser, T.D. and Hendriksen, N.B. Polymerase chain reaction assay for the detection of Bacillus cereus group cells. FEMS Microbiol. Lett. 202: 209-213 (2001)