Skip to main content
NCBI home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1996 Oct;34(10):2619–2622. doi: 10.1128/jcm.34.10.2619-2622.1996

Rapid identification of Salmonella serovars in feces by specific detection of virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination assay.

C H Chiu 1, J T Ou 1
PMCID: PMC229337  PMID: 8880536

Abstract

In order to make a rapid and definite diagnosis of Salmonella enteritis in children, an enrichment broth culture-multiplex PCR combination assay was devised to identify Salmonella serovars directly from fecal samples. Two pairs of oligonucleotide primers were prepared according to the sequences of the chromosomal invA and plasmid spvC genes. PCR with these two primers would produce either one amplicon (from the invA gene) or two amplicons (from the invA and spvC genes), depending on whether or not the Salmonella bacteria contained a virulence plasmid. The fecal sample was diluted 10- to 20-fold into gram-negative enrichment broth and incubated to eliminate inhibitory compounds and also to allow selective enrichment of the bacteria. One or two amplicons were obtained, the expected result if Salmonella bacteria were present. The detection limit of this PCR was about 200 bacteria per reaction mixture. The primers were specific, as no amplification products were obtained with 18 species and 22 isolates of non-Salmonella bacteria tested which could be present in the feces or cause contamination. In contrast, when 23 commonly seen Salmonella serovars (38 isolates) were tested, all were shown to carry the invA gene and seven concomitantly harbored the spvC gene of the virulence plasmid. This assay was applied to the diagnosis of Salmonella enteritis in 57 children who were suffering from mucoid and/or bloody diarrhea. Of the 57 children, 38 were PCR positive and 22 were culture positive. There were two culture-positive samples that were not detected by PCR. Thus, this PCR assay showed an efficiency of 95% (38 of 40), which is much higher than the 60% (24 of 40) by culture alone. Not only is this method more sensitive, rapid, and efficient but it will cause only an incremental increase in the cost of stool processing, since enrichment cultivation of fecal samples from diarrheal patients using gram-negative enrichment broth is a routine practice for identification in many diagnostic microbiology laboratories. This PCR method, therefore, has clinical application.

Full Text

The Full Text of this article is available as a PDF (175.0 KB).

Selected References

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

  1. Araj G. F., Chugh T. D. Detection of Salmonella spp. in clinical specimens by capture enzyme-linked immunosorbent assay. J Clin Microbiol. 1987 Nov;25(11):2150–2153. doi: 10.1128/jcm.25.11.2150-2153.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aspinall S. T., Hindle M. A., Hutchinson D. N. Improved isolation of salmonellae from faeces using a semisolid Rappaport-Vassiliadis medium. Eur J Clin Microbiol Infect Dis. 1992 Oct;11(10):936–939. doi: 10.1007/BF01962379. [DOI] [PubMed] [Google Scholar]
  3. Frankel G., Riley L., Giron J. A., Valmassoi J., Friedmann A., Strockbine N., Falkow S., Schoolnik G. K. Detection of Shigella in feces using DNA amplification. J Infect Dis. 1990 Jun;161(6):1252–1256. doi: 10.1093/infdis/161.6.1252. [DOI] [PubMed] [Google Scholar]
  4. Galán J. E., Curtiss R., 3rd Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6383–6387. doi: 10.1073/pnas.86.16.6383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Guiney D. G., Fang F. C., Krause M., Libby S., Buchmeier N. A., Fierer J. Biology and clinical significance of virulence plasmids in Salmonella serovars. Clin Infect Dis. 1995 Oct;21 (Suppl 2):S146–S151. doi: 10.1093/clinids/21.supplement_2.s146. [DOI] [PubMed] [Google Scholar]
  6. Gulig P. A., Caldwell A. L., Chiodo V. A. Identification, genetic analysis and DNA sequence of a 7.8-kb virulence region of the Salmonella typhimurium virulence plasmid. Mol Microbiol. 1992 May;6(10):1395–1411. doi: 10.1111/j.1365-2958.1992.tb00860.x. [DOI] [PubMed] [Google Scholar]
  7. Gulig P. A., Danbara H., Guiney D. G., Lax A. J., Norel F., Rhen M. Molecular analysis of spv virulence genes of the Salmonella virulence plasmids. Mol Microbiol. 1993 Mar;7(6):825–830. doi: 10.1111/j.1365-2958.1993.tb01172.x. [DOI] [PubMed] [Google Scholar]
  8. Kado C. I., Liu S. T. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol. 1981 Mar;145(3):1365–1373. doi: 10.1128/jb.145.3.1365-1373.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kongmuang U., Luk J. M., Lindberg A. A. Comparison of three stool-processing methods for detection of Salmonella serogroups B, C2, and D by PCR. J Clin Microbiol. 1994 Dec;32(12):3072–3074. doi: 10.1128/jcm.32.12.3072-3074.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ou J. T., Baron L. S., Dai X. Y., Life C. A. The virulence plasmids of Salmonella serovars typhimurium, choleraesuis, dublin, and enteritidis, and the cryptic plasmids of Salmonella serovars copenhagen and sendai belong to the same incompatibility group, but not those of Salmonella serovars durban, gallinarum, give, infantis and pullorum. Microb Pathog. 1990 Feb;8(2):101–107. doi: 10.1016/0882-4010(90)90074-z. [DOI] [PubMed] [Google Scholar]
  11. Rahn K., De Grandis S. A., Clarke R. C., McEwen S. A., Galán J. E., Ginocchio C., Curtiss R., 3rd, Gyles C. L. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Mol Cell Probes. 1992 Aug;6(4):271–279. doi: 10.1016/0890-8508(92)90002-f. [DOI] [PubMed] [Google Scholar]
  12. Ruiz J., Nunez M. L., Diaz J., Lorente I., Perez J., Gomez J. Comparison of five plating media for isolation of Salmonella species from human stools. J Clin Microbiol. 1996 Mar;34(3):686–688. doi: 10.1128/jcm.34.3.686-688.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  14. Stone G. G., Oberst R. D., Hays M. P., McVey S., Chengappa M. M. Detection of Salmonella serovars from clinical samples by enrichment broth cultivation-PCR procedure. J Clin Microbiol. 1994 Jul;32(7):1742–1749. doi: 10.1128/jcm.32.7.1742-1749.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Tada J., Ohashi T., Nishimura N., Shirasaki Y., Ozaki H., Fukushima S., Takano J., Nishibuchi M., Takeda Y. Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction. Mol Cell Probes. 1992 Dec;6(6):477–487. doi: 10.1016/0890-8508(92)90044-x. [DOI] [PubMed] [Google Scholar]
  16. Widjojoatmodjo M. N., Fluit A. C., Torensma R., Verdonk G. P., Verhoef J. The magnetic immuno polymerase chain reaction assay for direct detection of salmonellae in fecal samples. J Clin Microbiol. 1992 Dec;30(12):3195–3199. doi: 10.1128/jcm.30.12.3195-3199.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wilde J., Eiden J., Yolken R. Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions. J Clin Microbiol. 1990 Jun;28(6):1300–1307. doi: 10.1128/jcm.28.6.1300-1307.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Williamson C. M., Baird G. D., Manning E. J. A common virulence region on plasmids from eleven serotypes of Salmonella. J Gen Microbiol. 1988 Apr;134(4):975–982. doi: 10.1099/00221287-134-4-975. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES