Food Science of Animal Resources (한국축산식품학회지)

Korean Society for Food Science of Animal Resources (한국축산식품학회)
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Improved Detection of Viable Escherichia coli O157:H7 in Milk by Using Reverse Transcriptase-PCR

  • Choi, Suk-Ho (Division of Animal Resources and Life Science, Sangji University) ;
  • Lee, Seung-Bae (Division of Animal Resources and Life Science, Sangji University)
  • Received : 2010.06.16
  • Accepted : 2010.12.07
  • Published : 2011.04.30
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Abstract

A sensitive reverse transcriptase-PCR (RT-PCR) method to detect viable Escherichia coli O157:H7 in milk was established. The primer sets were designed based on the nucleotide sequences of the rfbE (per) and wbdN genes in the O157 antigen gene cluster of E. coli O157:H7. RT-PCR using five different primer sets yielded DNA with sizes of 655, 518, 450, and 149-bp, respectively. All five of the E. coli O157:H7 strains were detected by RT-PCR, but 11 other bacterial species were not. The sensitivity of RT-PCR was improved by adding yeast tRNA as a carrier to the crude RNA extract. The RT-PCR amplifying the 149-bp DNA fragment was the most sensitive for detecting E. coli O157:H7 and the most refractory to the bactericidal treatments. Heat treatment at $65^{\circ}C$ for 30 min was the least inhibitory of all bactericidal treatments. Treatment with RNase A strongly inhibited the RT-PCR of heated milk but not unheated milk. This study described RT-PCR methods that are specific and sensitive with a detection limit of 10 E. coli O157:H7 cells, and showed that pre-treating milk samples with RNase A improved the specificity to detect viable bacteria by RT-PCR.

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References

  1. Bickley, J., Short, J., McDowell, D., and Parks, H. (1996) Polymerase chain reaction (PCR) detection of Listeria monocytogenes in diluted milk and reversal of PCR inhibition caused by calcium ions. Lett. Appl. Microbiol. 22, 153-158. https://doi.org/10.1111/j.1472-765X.1996.tb01131.x
  2. Bilge, S. S., Vary, J. C. Jr., Dowell, S. F., and Tarr, P. I. (1996) Role of the Escherichia coli O157:H7 O side chain in adherence and analysis of rfb locus. Infect. Immun. 64, 4795-4801.
  3. de Wet, S. S., Denman, S. E., Sly, L, and McSweeney, C. S. (2008) An improved method for RNA extraction from carcass samples for detection of viable Escherchia coli O157:H7 by reverse-transcriptase polymerase chain reaction. Lett. Appl. Microbiol. 47, 399-404. https://doi.org/10.1111/j.1472-765X.2008.02462.x
  4. Dineen, S., Takeuchi, K., Soudah, J., and Boor, K. (1998) Persistence of Escherichia coli O157:H7 in dairy fermentation systems. J. Food Prot. 61, 1602-1608.
  5. Doyle, M. T., Zhao, T., Meng, J., and Zhao, S. (1997) Escherichia coli O157:H7. In: Food microbiology: fundamentals and frontiers, 5th ed, Doyle, M. P. (ed.), ASM Press, Washington, DC, pp. 171-191.
  6. Dupray, E., Caprais, M. P., Derrien, A., and Fach, P. (1997) Salmonella DNA persistence in natural seawaters using PCR analysis. J. Appl. Microbiol. 82, 507-510. https://doi.org/10.1046/j.1365-2672.1997.00143.x
  7. Fratamico, P. M., Sackitey, K., Wiedman, M., and Deng, M. Y. (1995) Detection of Escherichia coli O157:H7 by multiplex PCR. J. Clin. Microbiol. 33, 2188-2191.
  8. Gooding, C. M. and Choudary, P. V. (1997) Rapid and sensitive immunomagnetic separation-polymerase chain reaction method for the detection of Escherichia coli O157:H7 in raw milk and ice-cream. J. Dairy Res. 64, 87-93. https://doi.org/10.1017/S0022029996001896
  9. Griffin, P. M. and Tauxe, R. V. (1991) The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol. Rev. 13, 60-98.
  10. Hancock, D., Besser, T. E., Kinsel, M. L., Tarr, P. I., Rice, D. H., and Paros, M.G. (1994) The prevalence of Escherichia coli O157:H7 in dairy and beef cattle in Washington State. Epidemiol. Infect. 113, 199-207. https://doi.org/10.1017/S0950268800051633
  11. Herman, L. (1997) Detection of viable and dead Listeria monocytogenes by PCR. Food Microbiol. 14, 103-110. https://doi.org/10.1006/fmic.1996.0077
  12. Karmali, M. A. (1989) Infection by verocytotoxin-producing Escherchia coli. Clin. Microbiol. Rev. 2, 15-58.
  13. Klein, P. G. and Juneja V. K. (1997) Sensitive detection of viable Listeria monocytogenes by reverse transcription-PCR. Appl. Environ. Microbiol. 63, 4441-4448.
  14. Kushner, S. R. (1996) mRNA decay. In: Escherichia coli and Salmonella cellular and molecular Biology. Neidhart, F. C. (ed.), ASM Press, Washington, DC, pp. 849-860.
  15. Masters, C. L., Shallcross, J. A., and Mackey, B. M. (1994) Effect of stress treatments on the detection of Listeria monocytogenes and enterotoxigenic Escherichia coli by the polymerase chain reaction. J. Appl. Bacteriol. 77, 73-79. https://doi.org/10.1111/j.1365-2672.1994.tb03047.x
  16. McIngvale, S. C., Elhanafi, E., and Drake, M. A. (2002) Optimization of reverse transcriptase PCR to detect viable Shiga-toxin-producing Escherichia coli. Appl. Environm. Microbiol. 68, 799-806. https://doi.org/10.1128/AEM.68.2.799-806.2002
  17. McIngvale, S. C., Chen, X., McKillip, J. L., and Drake, M. A. (2000) Survival of Escherichia. coli O157:H7 in buttermilk as affected by contamination point and strorage temperature. J. Food Prot. 63, 441-444.
  18. McKillip, J. L., Jaykus, L. A., and Drake, M. (1998) rRNA stability in heat-killed and UV-irradiated enterotoxigenic Staphylococcus aureus and Escherichia coli O157:H7. Appl. Envion. Microb. 64, 4264-4268.
  19. Paton, A. W. and Paton, J. C. (1998) Detection and characterization of shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, $rfb_{011}$, and $rfb_{0157}$. J. Clin. Microbiol. 36, 598-602.
  20. Sails, A. D., Bolton, F. J., Fox, A. J., Wareing, D. R., and Greenway. D. L. (1998) A reverse transcriptase polymerase chain reaction assay for the detection of thermophilic Campylobacter spp. Mol. Cell Probes 12, 317-322 https://doi.org/10.1006/mcpr.1998.0184
  21. Sharma, V. J. (2006) Real-time reverse transcription-multiplex PCR for simultaneous and specific detection of rfbE and eae of genes of Escherichia coli O157:H7. Mol. Cell. Probes 20, 298-306. https://doi.org/10.1016/j.mcp.2006.03.001
  22. Sheridan, G. E., Szabo, E. A., and Mackey, B. M. (1999) Effect of post-treatment holding conditions on detection of tufA mRNA in ethanol-treated Escherichia coli: implications for RT-PCR-based indirect viability tests. Lett. Appl. Microb. 29, 375-379. https://doi.org/10.1046/j.1472-765X.1999.00644.x
  23. Szabo, E. A. and Mackey, B. M. (1999) Detection of Salmonella enteritidis by reverse transcriptase-polymerase chain reaction (PCR). Inter. J. Food Microbiol. 51, 113-122. https://doi.org/10.1016/S0168-1605(99)00106-3
  24. Szabo, R., Todd, E., and Jean, A. (1986) Method to isolate Escherichia coli O157:H7 from food. J. Food Prot. 49, 768-772.
  25. Vaitilingom, M., Gender, F., and Brignon, P. (1998) Direct detection of viable bacteria, molds, and yeasts by reverse transcriptase PCR in contaminated milk samples after heat treatment. Appl. Environ. Microbiol. 64, 1157-1160.
  26. Weeratna, R. D. and Doyle, M. (1991) Detection and production of verotoxin I of Escherichia coli O157:H7 in food. Appl. Environ. Microb. 57, 2951-2955.
  27. Witham, P. K., Yamashiro, C. T., Livak, K. J., and Batt, C.A. (1996) A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef. Appl. Environ. Microb. 62, 1347-1353.
  28. Yaron, S. and Matthew, K. R. (2002) A reverse transcriptasepolymerase chain reaction assay for detection of viable Escherichia coli O157:H7: investigation of specific target genes. J. Appl. Microbiol. 92, 633-640. https://doi.org/10.1046/j.1365-2672.2002.01563.x
  29. Yu, J. and Kaper, J. B. (1992) Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157:H7. Mol. Microbiol. 6, 411-417. https://doi.org/10.1111/j.1365-2958.1992.tb01484.x

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