Development of Differential Media and Multiplex PCR Assays for the Rapid Detection of Listeria monocytogenes

Listeria monocytogenes의 신속검출을 위한 선택배지 및 multiplex PCR 기법 개발

  • 정병열 (국립수의과학검역원) ;
  • 임현숙 (대구광역시 보건환경연구원) ;
  • 정석찬 (국립수의과학검역원)
  • Accepted : 2003.04.30
  • Published : 2003.06.30

Abstract

Listeria (L.) monocytogenes in samples could not be detected occasioally by faster growth of other Listeria spp. especially L. innocua. The aim of this study was to develop the differential media and multiplex polymerase chain reaction (PCR) assays for the rapid detection of L. monocytogenes. L. monocytogenes colonies were characterized by their ${\beta}$-hemolysis with fluorescence under 366 nm UV light on the Listeria hemolysis agar (LHA). L. innocua, a species commonly present in foods, did not produce ${\beta}$-hemolysis on LHA. Therefore, one or more colonies of L. monocytogenes were easily distinguished from large populations of L. innocua. The multiplex PCR assays were developed to distinguish from L. monocytogenes and other Listeria spp. with two pairs of primers. The primers were designed in 16S rRNA and listeriolysin O gene for specific amplification of all members of the genus Listeria and L. monocytogenes, respectively. The multiplex PCR assays produced 560 and 938 bp products in L. monocytogenes; only 938 bp products in the genus Listeria. The multiplex PCR assays could detect as little as 50 pg of L monocytogenes DNA. These results indicated that the differential media and multiplex PCR assays might be useful diagnostic tools for the rapid detection of L. monocytogenes.

References

  1. Bansal, N. S. Development of a polymerase chain reaction assay for the detection of Listeria monocytogenes in foods. Lett. Appl. Microbiol. 1996, 22, 353-356
  2. Bej, A. K., DiCesare, J. L., Haff, L. and Atlas, R. M. Detection of Escherichia coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid. Appl. Environ. Microbiol. 1991, 57, 1013-1017
  3. Bessesen, M. T., Luo, Q., Rotbart, A., Blaser, M. J. and Ellison III, R. T. Detection of Listeria monocytogenes by using the polymerase chain reaction. Appl. Environ. Microbiol. 1990, 56, 2930-2932
  4. Beumer, R. R. and Brinkman, E. Detection of Listeria spp. with a monoclonal enzyme-linked immunosorbant assay (ELISA). Food Microbiol. 1989, 6, 171-177
  5. Beumer, R. R., te Giffel, M. C. and Cox, L. J. Optimization of haemolysis in enhanced haemolysis agar (EHA)-a selective medium for the isolation of Listeria monocytogenes. Lett. Appl. Microbiol. 1997, 24, 421-425
  6. Beumer, R. R., te Giffel, M. C., Kok, M. T. C. and Rombouts, F. M, Confirmation ald identification of Listeria spp. Lett. Appl. Microbiol. 1996, 22, 448-452
  7. Cox, L. J., Siebenga, A., Pedrazzini, C., and Moreton, J. Enhanced haemolytic agar(EHA)-an improved selective and differential medium for isolation of Listeria monocytogenes. Food Microbiol. 1991, 8, 37-49
  8. Farber, J. M and Peterkin, P. L Listeria momcytogenes, a food-borme pathogen. Microbiol. Rev. 1991, 55, 476-511
  9. Golsteyn Thomas, E. J., King, R. K., Burchak, J. and Gannon, V. P. J. Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction. Appl. Environ Microbiol. 1991, 57, 2576-2580
  10. Johansson, T. Enhanced detection and enumeration of Listeria monocytogenes from foodstuffs and food-processing environments, Int. J. Food Microbiol. 1998, 40, 77-85
  11. Lane, D. J., Pace, B., Olsen, G. J., Stahl, D. A., Sogin, M. L. and Pace, N. R. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc. Nat. Aca. Sci. USA. 1985, 82, 6955-6959
  12. McClain, D. and Lee, W. H. Development of USDAFSIS method for isolation of Listeria monocytogenes from raw meat and poultry. J. Assoc. Off. Anal. Chem. 1988, 71, 660-664
  13. McDonald, F. and Sutherland, A. D. Important differences between the generation times of Listeria monocytogenes and Listeria innocua in two Listeria enrichment broth. J. Dairy Res. 1994, 61, 433-436
  14. Poysky, F. T., Paranjpye, R. N., Lashbrook, L. C., Peterson, M. E., Perloy, G. A. and Eklund, M. W. Selective and differential medium for isolation of Listeria monocytogenes from fcods. J. Food Prot. 1993, 56, 326-329
  15. Rocourt, J., Boerlin, P., Grimont, F., Jacquet, C. and Piffaretti, J. C Assignment of Listeria grayi and Listeria murrayi to a single species, Listeria grayi, with a revised description of Listeria grayi. Int. J. Syst. Bacteriol. 1992, 42, 171-174
  16. Stackenbrandt, E. and Curiale, M. Detection of Listeria. European Patent Application 88308820.5. 1988
  17. Skovgaard, N. and Morgan, C. A. Detection of Listeria spp in feces from animals, in feeds, and in raw foods of animal origin. Int. J. Food Microbiol. 1988, 6, 229-242
  18. Warburton, D. W., Farber, J. M., Armstrong, A., Caldeira, R., Hunt, T., Messier, S., Plante, R., Tiwari, N. P. and Vinet, J. A comparative study of the 'FDA' and 'USDA' methods for the detecdon of Listeria monocytogenes in foods. Int. J. Food Microbiol. 1991, 13, 105-118
  19. Wernars, K., Heuvelman, C. J., Chakraborty, T. and Notermans, S. H. Use of the polymerase chain reaction for direct detection of Ltsteria monocytogenes in soft cheese. J. Appl. Bacteriol. 1991, 70, 121-126