Journal of Microbiology

The Microbiological Society of Korea (한국미생물학회)
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PCR Method Based on the ogdH Gene for the Detection of Salmonella spp. from Chicken Meat Samples

  • Jin, Un-Ho (Department of Biochemistry and Molecular Biology, College of Oriental Medicine, Dongguk University and NRL-Glycobiology) ;
  • Cho, Sung-Hak (Laboratory of Enteric Infections, National Institute of Health) ;
  • Kim, Min-Gon (Korea Research Institute of Bioscience and Biotechnology) ;
  • Ha, Sang-Do (Department of Food Science & Technology, Chung-Ang University) ;
  • Kim, Keun-Sung (Department of Food Science & Technology, Chung-Ang University) ;
  • Lee, Kyu-Ho (Environmental Science, Hankuk University of Foreign Studies) ;
  • Kim, Kwang-Yup (Department of Food Science & Technology, Chungbuk National University) ;
  • Chung, Duck Hwa (Division of Applied Life Science, Gyeongsang National University) ;
  • Lee, Young-Choon (Faculty of Biotechnology, Dong-A University) ;
  • Kim, Cheorl-Ho (Department of Biochemistry and Molecular Biology, College of Oriental Medicine, Dongguk University and NRL-Glycobiology)
  • Published : 2004.09.01
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Abstract

In a previous paper, the ogdH gene that encodes 2-oxoglutarat dehydrogenase was isolated from Salmonella typhimurium. The catalytic N-terminal region in the enzyme was found to be very specific for the Salmonella species. Therefore, the aim of the present study was to detect S. typhimurium in food sources using primers designed for OGDH-l and OGDH-2 which were based on the salmonella-specific region of the ogdH gene. A simple polymerase chain reaction (PCR) detection method was developed to detect low numbers of S. typhimurium in a chicken meat microbial consortium. Using the ogdH-specific primers under stringent amplification conditions and for gene probe analysis, fewer than 100 colony-forming units (CFUs) were detectable when pure cultures were employed. When the PCR assay was run on S. typhimurium-contaminated meat contents, only the positive meat samples containing as few as 200 CFUs reacted to the assay. The method employed for sample processing is simple and it was determined to provide a sensitive means of detecting trace amounts of S. typhimurium-specific sequences in the presence of mixed meat microbial populations. When compared with six representative intestinal gram-negative bacterial strains in foods, including Vibrio parahaemolyticus, V. vulnificus, Enterobacter cloacae, E. coli O157:H7, Pseudomonas aeruginosa, and Proteus sp., S. typhimurium had a unique and distinct PCR product (796 bp). In conclusion, the two OGDH primers were found to be rapid and sensitive detectors of Salmonella spp for the PCR method.

Keywords

References

  1. Bej, A.K., M.H. Mahbubani, M.J. Boyce, and R.M. Atlas. 1994. Detection of Salmonella spp. in oysters by PCR. Appl. Environ. Microbiol. 60, 368-373
  2. D'Aoust, J. 1985. Infective dose of Salmonella typhimurium in cheddar cheese. Am. J. Epidemiol. 122, 717-720
  3. Darlison, M.G., M.E. Spencer, and J.R. Guest. 1984. Nucleotide sequence of the sucA gene encoding the 2-oxoglutarate dehydrogenase of Escherichia coli K12. Eur. J. Biochem. 141, 351-359
  4. Dodd, C.E.R., G.A.B. Stewart, and W.M. Waltes. 1990. Biotechnology-based methods for the detection, enumeration and epidemiology of food poisoning and spoilage organisms. Biotechnol. Gent. Eng. Rev. 8, 1-52
  5. Ezpuerra, E., A. Burnens, C. Jones, and J. Stansey. 1993. Genotypic typing and phylogenetic analysis of Salmonella paratyphi B and S. iaua with IS200. J. Gen. Microbiol. 139, 2409-2414
  6. Fitts, R. 1985. Development of a DNA hybridization test for the presence of Salmonella in foods. Food Technol. 39, 95-102
  7. Greenwood, M. and W. Hooper. 1983. Chocolate bars contaminated with Salmonella napoli: an infective study. Br. Med. J. 286, 1394-1397
  8. Hill, W.E., S.P. Stacey, M.W. Trucksess, P. Feng, C.A. Kaysner, and K.A Lampel. 1991. Polymerase chain reaction identification of Vibrio vulnifcus in artificially contaminated oysters. Appl. Environ. Microbiol. 57, 707-711
  9. Jin, U.H., T.W. Chung, J.K. Kim, K.S. Nam, S.D. Ha, and C.H. Kim. 1999. Differentiation of Salmonella typhimurium from gram-negative intestinal microbes by randomly amplified polymorphic DNA (RAPD) figerprinting. J. Microbiol. 38(1), 8-10
  10. Jin, U.H., T.W. Chung, Y.C. Lee, S.D. Ha, and C.H. Kim. 2001. Molecular cloning and functional expression of the rfaE gene required for lipopolysaccharide biosynthesis in Salmonella typhimurium. Glycoconjugate J. 18, 779-787.
  11. Kim C.H. 2003. Catalytic domain of Salmonella typhimurium 2-oxoglutarate dehydrogenase is localized in N-terminal region. J. Molecular Catalysis B: Enzymatic. 26, 193-200
  12. Knight, I.T., S. Shults, C.W. Kasper, and R.R. Colwell. 1990. Direct detection Salmonella spp. in estuaries by using a DNA probe. Appl. Environ. Microbiol. 56, 1059-1066
  13. Koch, W.H., W.I. Payne, B.A. Wentz, and T.A. Cebula. 1993. Rapid polymerase chain reaction method for detection of Vibrio cholerae in foods. Appl. Env. Microbiol. 59, 556-560
  14. Maniatis, T., E.F. Fritsch, and J. Sambrook, 1982. Molecular cloning, a laboratory manual. Cold Spring Harbour Laboratory, NY
  15. Olsen, J.E., S. Aabo, W. Hill, S. Notermans, K. Wernars, P.E. Ganum, T. Popovic, H.N. Rasmussen, and O. Slavik. 1995. Probes and polymerase chain reaction for detection of foodborne bacterial pathogens. Int. J. Food Microbiol. 28, 1-78
  16. Pettit, F.H., L. Hamilton, P. Munk, G. Namihira, M.H. Eley, C.R. Willms, and L.J. Reed. 1973. $\alpha$-Keto acid dehydrogenase complexes. XIX. Subunit structure of the Escherichia coli $\alpha$-ketoglutarate dehydrogenase complex. J. Biol. Chem. 248, 5282-5290.
  17. Pillai, S.D., S.C. Ricke, D.J. Nisbet, D.E. Corrier and J.R. DeLoach. 1994. A Rapid method for screening for Salmonella typhimurium in a chiken cecal microbial consortium using gene amplification. Avian Dis. 38, 558-604
  18. Ricaud, P.M., M.J. Howard, E.L. Roberts, R.W. Broadhurst, and R.N. Perham. 1996. Three-dimensional structure of the lipoil domain from the dihydrolipoil succinyltransferase component of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli. J. Mol. Biol. 264,179-190
  19. Shin, J.W., J.K. Kang, K.I. Jang, and K.Y. Kim. 2002. Intestinal colonization characteristics of Lactobacillus spp. isolated from chicken cecum and competitive inhibition against Salmonella typhimurium. J. Microbiol. Biotechnol. 12, 576-569
  20. Shin, S.Y., J.H. Park, and W.J. Kim. 1999. Specific detection of enteropathogen Campylobacter jejuni in food using a polymerase chain reaction. J. Microbiol. Biotechnol. 9, 184-190
  21. Sommer, R., and D. Tautz. 1989. Minimal homology requirement for PCR primers. Nucleic Acids Res. 17, 6749
  22. Won, M.S., S.Y. Kim, S.H. Lee, C.J. Kim, H.S. Kim, M.H. Jun, and K.B. Song. 2001. Prediction of the scondary structure of the Agf A subunit of Salmonella enteritidis overexpressed as an MBP-fused protein. J. Microbiol. Biotechnol. 11, 164-166