Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1991 Sep;57(9):2576–2580. doi: 10.1128/aem.57.9.2576-2580.1991

Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction.

E J Thomas 1, R K King 1, J Burchak 1, V P Gannon 1
PMCID: PMC183622  PMID: 1768130

Abstract

A sensitive and specific method for detection of Listeria monocytogenes in milk and ground-beef samples is described. It consists of culturing samples in listeria enrichment broth (LEB) and subculturing them from LEB to listeria plating media, followed by DNA extraction and species-specific detection of the organism by using the polymerase chain reaction (PCR). In developing the L. monocytogenes PCR assay, five oligonucleotide primers complementary to the nucleotide sequence of the listeriolysin O gene were synthesized and used in amplification experiments. PCR products of the predicted size, based on nucleotide sequence information, were generated with DNA from all of 72 L. monocytogenes strains with five different primer pairs. DNA from Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeia murrayi strains and a panel of 47 bacterial strains representing 17 genera did not generate PCR products with the primer pairs employed. As little as 1 pg of L. monocytogenes DNA could be detected with the assay. To determine the most sensitive culture protocol to use in conjunction with the PCR assay, milk (10 ml) and ground-beef (25 g) samples were inoculated with L. monocytogenes at concentrations ranging from 0 to 10(5) CFU ml-1 or g-1, as appropriate for the sample. PCR assays on DNA extracted from growth on listeria plating media, inoculated with 24-h LEB samples cultures, were most sensitive, allowing detection of as little as 0.1 CFU of L. monocytogenes ml-1 or g-1 of milk and ground beef, respectively.

Full text

PDF
2576

Images in this article

2578Image
on p.2578
2578Image
on p.2578
2578Image
on p.2578
2579Image
on p.2579

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bessesen M. T., Luo Q. A., Rotbart H. A., Blaser M. J., Ellison R. T., 3rd Detection of Listeria monocytogenes by using the polymerase chain reaction. Appl Environ Microbiol. 1990 Sep;56(9):2930–2932. doi: 10.1128/aem.56.9.2930-2932.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Border P. M., Howard J. J., Plastow G. S., Siggens K. W. Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction. Lett Appl Microbiol. 1990 Sep;11(3):158–162. doi: 10.1111/j.1472-765x.1990.tb00149.x. [DOI] [PubMed] [Google Scholar]
  3. Cossart P., Vicente M. F., Mengaud J., Baquero F., Perez-Diaz J. C., Berche P. Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation. Infect Immun. 1989 Nov;57(11):3629–3636. doi: 10.1128/iai.57.11.3629-3636.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Datta A. R., Wentz B. A., Hill W. E. Detection of hemolytic Listeria monocytogenes by using DNA colony hybridization. Appl Environ Microbiol. 1987 Sep;53(9):2256–2259. doi: 10.1128/aem.53.9.2256-2259.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Datta A. R., Wentz B. A., Russell J. Cloning of the listeriolysin O gene and development of specific gene probes for Listeria monocytogenes. Appl Environ Microbiol. 1990 Dec;56(12):3874–3877. doi: 10.1128/aem.56.12.3874-3877.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Datta A. R., Wentz B. A., Shook D., Trucksess M. W. Synthetic oligodeoxyribonucleotide probes for detection of Listeria monocytogenes. Appl Environ Microbiol. 1988 Dec;54(12):2933–2937. doi: 10.1128/aem.54.12.2933-2937.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Deneer H. G., Boychuk I. Species-specific detection of Listeria monocytogenes by DNA amplification. Appl Environ Microbiol. 1991 Feb;57(2):606–609. doi: 10.1128/aem.57.2.606-609.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Farber J. M., Losos J. Z. Listeria monocytogenes: a foodborne pathogen. CMAJ. 1988 Mar 1;138(5):413–418. [PMC free article] [PubMed] [Google Scholar]
  9. Flamm R. K., Hinrichs D. J., Thomashow M. F. Cloning of a gene encoding a major secreted polypeptide of Listeria monocytogenes and its potential use as a species-specific probe. Appl Environ Microbiol. 1989 Sep;55(9):2251–2256. doi: 10.1128/aem.55.9.2251-2256.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fleming D. W., Cochi S. L., MacDonald K. L., Brondum J., Hayes P. S., Plikaytis B. D., Holmes M. B., Audurier A., Broome C. V., Reingold A. L. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N Engl J Med. 1985 Feb 14;312(7):404–407. doi: 10.1056/NEJM198502143120704. [DOI] [PubMed] [Google Scholar]
  11. Gaillard J. L., Berche P., Sansonetti P. Transposon mutagenesis as a tool to study the role of hemolysin in the virulence of Listeria monocytogenes. Infect Immun. 1986 Apr;52(1):50–55. doi: 10.1128/iai.52.1.50-55.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gray M. L., Killinger A. H. Listeria monocytogenes and listeric infections. Bacteriol Rev. 1966 Jun;30(2):309–382. doi: 10.1128/br.30.2.309-382.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Groves R. D., Welshimer H. J. Separation of pathogenic from apathogenic Listeria monocytogenes by three in vitro reactions. J Clin Microbiol. 1977 Jun;5(6):559–563. doi: 10.1128/jcm.5.6.559-563.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. JENKINS E. M., NJOKU-OBI A. N., ADAMS E. W. PURIFICATION OF THE SOLUBLE HEMOLYSINS OF LISTERIA MONOCYTOGENES. J Bacteriol. 1964 Aug;88:418–424. doi: 10.1128/jb.88.2.418-424.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kathariou S., Metz P., Hof H., Goebel W. Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes. J Bacteriol. 1987 Mar;169(3):1291–1297. doi: 10.1128/jb.169.3.1291-1297.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kim C. M., Graves L. M., Swaminathan B., Mayer L. W., Weaver R. E. Evaluation of hybridization characteristics of a cloned pRF106 probe for Listeria monocytogenes detection and development of a nonisotopic colony hybridization assay. Appl Environ Microbiol. 1991 Jan;57(1):289–294. doi: 10.1128/aem.57.1.289-294.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. King W., Raposa S., Warshaw J., Johnson A., Halbert D., Klinger J. D. A new colorimetric nucleic acid hybridization assay for Listeria in foods. Int J Food Microbiol. 1989 Jun;8(3):225–232. doi: 10.1016/0168-1605(89)90017-2. [DOI] [PubMed] [Google Scholar]
  18. Köhler S., Leimeister-Wächter M., Chakraborty T., Lottspeich F., Goebel W. The gene coding for protein p60 of Listeria monocytogenes and its use as a specific probe for Listeria monocytogenes. Infect Immun. 1990 Jun;58(6):1943–1950. doi: 10.1128/iai.58.6.1943-1950.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lachica R. V. Selective plating medium for quantitative recovery of food-borne Listeria monocytogenes. Appl Environ Microbiol. 1990 Jan;56(1):167–169. doi: 10.1128/aem.56.1.167-169.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lampel K. A., Jagow J. A., Trucksess M., Hill W. E. Polymerase chain reaction for detection of invasive Shigella flexneri in food. Appl Environ Microbiol. 1990 Jun;56(6):1536–1540. doi: 10.1128/aem.56.6.1536-1540.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Leimeister-Wächter M., Chakraborty T. Detection of listeriolysin, the thiol-dependent hemolysin in Listeria monocytogenes, Listeria ivanovii, and Listeria seeligeri. Infect Immun. 1989 Aug;57(8):2350–2357. doi: 10.1128/iai.57.8.2350-2357.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mengaud J., Chenevert J., Geoffroy C., Gaillard J. L., Cossart P. Identification of the structural gene encoding the SH-activated hemolysin of Listeria monocytogenes: listeriolysin O is homologous to streptolysin O and pneumolysin. Infect Immun. 1987 Dec;55(12):3225–3227. doi: 10.1128/iai.55.12.3225-3227.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mengaud J., Vicente M. F., Chenevert J., Pereira J. M., Geoffroy C., Gicquel-Sanzey B., Baquero F., Perez-Diaz J. C., Cossart P. Expression in Escherichia coli and sequence analysis of the listeriolysin O determinant of Listeria monocytogenes. Infect Immun. 1988 Apr;56(4):766–772. doi: 10.1128/iai.56.4.766-772.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Notermans S., Chakraborty T., Leimeister-Wächter M., Dufrenne J., Heuvelman K. J., Maas H., Jansen W., Wernars K., Guinee P. Specific gene probe for detection of biotyped and serotyped Listeria strains. Appl Environ Microbiol. 1989 Apr;55(4):902–906. doi: 10.1128/aem.55.4.902-906.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Peterkin P. I., Idziak E. S., Sharpe A. N. Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe. Appl Environ Microbiol. 1991 Feb;57(2):586–591. doi: 10.1128/aem.57.2.586-591.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Roll J. T., Czuprynski C. J. Hemolysin is required for extraintestinal dissemination of Listeria monocytogenes in intragastrically inoculated mice. Infect Immun. 1990 Sep;58(9):3147–3150. doi: 10.1128/iai.58.9.3147-3150.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schlech W. F., 3rd, Lavigne P. M., Bortolussi R. A., Allen A. C., Haldane E. V., Wort A. J., Hightower A. W., Johnson S. E., King S. H., Nicholls E. S. Epidemic listeriosis--evidence for transmission by food. N Engl J Med. 1983 Jan 27;308(4):203–206. doi: 10.1056/NEJM198301273080407. [DOI] [PubMed] [Google Scholar]
  28. Schwartz B., Hexter D., Broome C. V., Hightower A. W., Hirschhorn R. B., Porter J. D., Hayes P. S., Bibb W. F., Lorber B., Faris D. G. Investigation of an outbreak of listeriosis: new hypotheses for the etiology of epidemic Listeria monocytogenes infections. J Infect Dis. 1989 Apr;159(4):680–685. doi: 10.1093/infdis/159.4.680. [DOI] [PubMed] [Google Scholar]
  29. Wernars K., Heuvelman C. J., Chakraborty T., Notermans S. H. Use of the polymerase chain reaction for direct detection of Listeria monocytogenes in soft cheese. J Appl Bacteriol. 1991 Feb;70(2):121–126. doi: 10.1111/j.1365-2672.1991.tb04437.x. [DOI] [PubMed] [Google Scholar]
  30. Wesley I. V., Wesley R. D., Heisick J., Harrell F., Wagner D. Characterization of Listeria monocytogenes isolates by Southern blot hybridization. Vet Microbiol. 1990 Sep;24(3-4):341–353. doi: 10.1016/0378-1135(90)90182-u. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES