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. 1997 Oct;63(10):4127–4131. doi: 10.1128/aem.63.10.4127-4131.1997

A simple filtration technique to detect enterohemorrhagic Escherichia coli O157:H7 and its toxins in beef by multiplex PCR.

K Venkateswaran 1, Y Kamijoh 1, E Ohashi 1, H Nakanishi 1
PMCID: PMC168729  PMID: 9327582

Abstract

Primers, specific for a unique base substitution in uidA of Escherichia coli O157:H7, were coupled with oligonucleotides for the shiga-like toxin I (SLT-I) and SLT-II genes in a multiplex PCR assay. A minimum of 10(2) CFU per PCR (10 microliters) was necessary to amplify E. coli O157:H7-specific bands by multiplex PCR. Food particles as well as various unknown metabolic by-products of bacteria inhibited the PCR, but a simple two-step filtration procedure eliminated this inhibition. To reliably generate PCR products, an E. coli inoculum of 10(3) CFU g of food slurry-1 in a nonspecific medium was required with 6 h of enrichment at 37 degrees C. However, when the food homogenate was incubated overnight, E. coli O157:H7 at an initial inoculum of even 1 CFU g-1 was detected.

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Selected References

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  1. Aleksić S., Karch H., Bockemühl J. A biotyping scheme for Shiga-like (Vero) toxin-producing Escherichia coli O157 and a list of serological cross-reactions between O157 and other gram-negative bacteria. Zentralbl Bakteriol. 1992 Jan;276(2):221–230. doi: 10.1016/s0934-8840(11)80009-5. [DOI] [PubMed] [Google Scholar]
  2. Bej A. K., McCarty S. C., Atlas R. M. Detection of coliform bacteria and Escherichia coli by multiplex polymerase chain reaction: comparison with defined substrate and plating methods for water quality monitoring. Appl Environ Microbiol. 1991 Aug;57(8):2429–2432. doi: 10.1128/aem.57.8.2429-2432.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bettelheim K. A., Evangelidis H., Pearce J. L., Sowers E., Strockbine N. A. Isolation of a Citrobacter freundii strain which carries the Escherichia coli O157 antigen. J Clin Microbiol. 1993 Mar;31(3):760–761. doi: 10.1128/jcm.31.3.760-761.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cebula T. A., Payne W. L., Feng P. Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR. J Clin Microbiol. 1995 Jan;33(1):248–250. doi: 10.1128/jcm.33.1.248-250.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cha R. S., Zarbl H., Keohavong P., Thilly W. G. Mismatch amplification mutation assay (MAMA): application to the c-H-ras gene. PCR Methods Appl. 1992 Aug;2(1):14–20. doi: 10.1101/gr.2.1.14. [DOI] [PubMed] [Google Scholar]
  6. Chang G. W., Brill J., Lum R. Proportion of beta-D-glucuronidase-negative Escherichia coli in human fecal samples. Appl Environ Microbiol. 1989 Feb;55(2):335–339. doi: 10.1128/aem.55.2.335-339.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feng P. Identification of Escherichia coli serotype O157:H7 by DNA probe specific for an allele of uid A gene. Mol Cell Probes. 1993 Apr;7(2):151–154. doi: 10.1006/mcpr.1993.1021. [DOI] [PubMed] [Google Scholar]
  8. Feng P., Lampel K. A. Genetic analysis of uidA expression in enterohaemorrhagic Escherichia coli serotype O157:H7. Microbiology. 1994 Aug;140(Pt 8):2101–2107. doi: 10.1099/13500872-140-8-2101. [DOI] [PubMed] [Google Scholar]
  9. Feng P., Lum R., Chang G. W. Identification of uidA gene sequences in beta-D-glucuronidase-negative Escherichia coli. Appl Environ Microbiol. 1991 Jan;57(1):320–323. doi: 10.1128/aem.57.1.320-323.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fratamico P. M., Buchanan R. L., Cooke P. H. Virulence of an Escherichia coli O157:H7 sorbitol-positive mutant. Appl Environ Microbiol. 1993 Dec;59(12):4245–4252. doi: 10.1128/aem.59.12.4245-4252.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gannon V. P., King R. K., Kim J. Y., Thomas E. J. Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction. Appl Environ Microbiol. 1992 Dec;58(12):3809–3815. doi: 10.1128/aem.58.12.3809-3815.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Griffin P. M., Tauxe R. V. The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol Rev. 1991;13:60–98. doi: 10.1093/oxfordjournals.epirev.a036079. [DOI] [PubMed] [Google Scholar]
  13. Karmali M. A. Infection by verocytotoxin-producing Escherichia coli. Clin Microbiol Rev. 1989 Jan;2(1):15–38. doi: 10.1128/cmr.2.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lang A. L., Tsai Y. L., Mayer C. L., Patton K. C., Palmer C. J. Multiplex PCR for detection of the heat-labile toxin gene and shiga-like toxin I and II genes in Escherichia coli isolated from natural waters. Appl Environ Microbiol. 1994 Sep;60(9):3145–3149. doi: 10.1128/aem.60.9.3145-3149.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Levine M. M. Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J Infect Dis. 1987 Mar;155(3):377–389. doi: 10.1093/infdis/155.3.377. [DOI] [PubMed] [Google Scholar]
  16. O'Brien A. D., Holmes R. K. Shiga and Shiga-like toxins. Microbiol Rev. 1987 Jun;51(2):206–220. doi: 10.1128/mr.51.2.206-220.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Rice E. W., Sowers E. G., Johnson C. H., Dunnigan M. E., Strockbine N. A., Edberg S. C. Serological cross-reactions between Escherichia coli O157 and other species of the genus Escherichia. J Clin Microbiol. 1992 May;30(5):1315–1316. doi: 10.1128/jcm.30.5.1315-1316.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Saliba L. J., Helmer R. Health risks associated with pollution of coastal bathing waters. World Health Stat Q. 1990;43(3):177–187. [PubMed] [Google Scholar]
  19. Todd E. C., Szabo R. A., Peterkin P., Sharpe A. N., Parrington L., Bundle D., Gidney M. A., Perry M. B. Rapid hydrophobic grid membrane filter-enzyme-labeled antibody procedure for identification and enumeration of Escherichia coli O157 in foods. Appl Environ Microbiol. 1988 Oct;54(10):2536–2540. doi: 10.1128/aem.54.10.2536-2540.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Venkateswaran K., Murakoshi A., Satake M. Comparison of commercially available kits with standard methods for the detection of coliforms and Escherichia coli in foods. Appl Environ Microbiol. 1996 Jul;62(7):2236–2243. doi: 10.1128/aem.62.7.2236-2243.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Willshaw G. A., Smith H. R., Roberts D., Thirlwell J., Cheasty T., Rowe B. Examination of raw beef products for the presence of Vero cytotoxin producing Escherichia coli, particularly those of serogroup O157. J Appl Bacteriol. 1993 Nov;75(5):420–426. doi: 10.1111/j.1365-2672.1993.tb02797.x. [DOI] [PubMed] [Google Scholar]
  22. Witham P. K., Yamashiro C. T., Livak K. J., Batt C. A. A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef. Appl Environ Microbiol. 1996 Apr;62(4):1347–1353. doi: 10.1128/aem.62.4.1347-1353.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

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