BibTex RIS Cite

Comparison of real-time PCR and conventional cultural methods to detect Escherichia coli O157:H7 in spices in Turkey

Year 2012, Volume: 71 Issue: 2, 113 - 120, 25.12.2012

Abstract

In this study, 66 different spice samples were analyzed in order to compare Real-Time PCR and conventional
cultural methods for the detection of E. coli O157:H7 in spices. Thus, 66 different spice samples
(raw materials) were collected from various places in Izmir, Turkey. Conventional cultural methods
included pre-enrichment step, selection step and confirmation step. The molecular method included
two steps: pre-enrichment and Real-Time PCR step. Beside these methods, total aerobic mesophilic
bacteria, coliform, fecal coliform in samples, were also counted and the presence of Salmonella sp.
was investigated in order to determine total microbial load in samples. The results showed that aerobic
mesophilic bacteria counts were 5.1x103 -2.0x108 CFU/g, coliform counts were 8.0 x 102 CFU/g for the
average values of all samples. Salmonella sp. was not found in any of the samples.In four samples E.
coli O157:H7 positive by Real-Time PCR were found while all of the samples were negative by cultural
methods. We observed that the results of Real-Time PCR were more reliable than conventional methods.
Furthermore, the results were obtained in only 20 hours by Real-Time PCR method whereas conventional
cultural method was completed in 4 days.
Keywords: Cultural method, E. coli O157:H7, real-time PCR, spice

References

  • Akkaya L. and Alişarlı M. (2006) Afyonkarahisar’da tüketime sunulan peynirlerde Listeria mono- cytogenes ve Salmonella spp. varlığının be- lirlenmesi. Yüzüncü Yıl Üniversitesi Veteriner Fakültesi Dergisi, 17: 87-91.
  • Alişarlı M. and Akman HN. (2004) Perakende satılan kıymaların Escherichia coli O157:H7 yönünden incelenmesi. Yüzüncü Yıl Üniversitesi Veteriner Fakültesi Dergisi, 15: 65-69.
  • Ateş M., Özkızılcık A. and Tabakoğlu C. (2011) Microbiological Analysis of Stuffed Mussels Sold in The Streets. Indian Journal of Micro- biology, 51: 350-354.
  • Bhagwat A.A. (2002) Simultaneous detection of Escherichia coli O157:H7, Listeria monocyto- genes and Salmonella strains by real-time PCR. International Journal of Food Microbiology, 84: 217-224.
  • Catarame T.M.G., O’hanlon K.A., McDowell D.A., Blair I.S. and Duffy G. (2006) Comparison of a Real-Time Polymerase Reaction Assay with a cultural method for the detection of Salmonella in retail meat samples. Journal of Food Safety, 26: 1-15.
  • Chigbu P., Gordon S. and Strange R.T. (2005) Fe- cal coliform bacteria disapperance rates in a North-central Gulf of Mexico estuary. Estuarine, Coastal and Shelf Science, 65: 309-318.
  • Chui S. (2004) Detection and characterization of Esherichia coli O157:H7 and Salmonella in food, PhD, University of Maryland pp. 3.
  • Ellingson E.L.J., Koziczkowski J.J., Anderson L.J., Carlson A.S. and Sharma K.V. (2005) Rapid PCR detection of enterohemorrhagic Esch- erichia coli (EHEC) in bovine food products and feces. Molecular Cell Probes, 19: 213-217.
  • Espy M.J., Uhl J.R., Sloan L.M., Buckwalter S.P., Jones M.F., Vetter E.A., Yao J.D.C., Wengenack N.L. and Rosenblatt J.E. (2006) Real-Time PCR in clinical microbiology: Applications for rou- tine laboratory testing. Clinical Microbiology Reviews, 19: 165-256.
  • Feng P., Weagant S.D., Grant M.A. and Burkhardt W. (2002) BAM: Enumeration of Escherichia coli and the Coliform Bacteria. Bacteriologi- cal Analytical Manual 8th Edition, Revision A, Chapter 4.
  • Fitzmaurice J. (2008) Detection of verotoxin genes VT 1 and VT 2 in Escherichia coli O157:H7 in minced beef using Immunocapture and Real- Time PCR. In: Louise O’Connor (Ed.) Diag- nostic Bacteriology Protocols, New Zealand 345: 91-96.
  • Geissler K., Manafi M., Amoros I. and Alonso J.L. (2000) Quantitative determination of total co- liforms and Escherichia coli in marine waters with chromogenic and fluorogenic media. Jour- nal of Applied Microbiology 88: 280-285.
  • Islam A.M., Heuvelink A.E., Talıkder A.K. and Boer E.D. (2006) Immunoconcentration of shiga toxin-producing Escherichia coli O157 from animal faeces and raw meats by using dyna- beads anti-E. coli O157 and the VIDAS System. International Journal of Food Microbiology, 109: 151-156.
  • Kök F., Özbey G. and Muz A. (2007) Aydın İlinde Satışa Sunulan Fermente Sucukların Mikro- biyolojik Kalitelerinin İncelenmesi. Fırat Üni- versitesi Sağlık Bilimleri Dergisi, 21: 249-252.
  • Liu Y., Gilchrist A., Zhang J., and Li XF. (2008). Detection of Viable but Nonculturable Esch- erichia coli O157:H7 Bacteria in Drinking Water and River Water. Applied and Environmental Microbiology 74: 1502-1507.
  • Nataro J.P. and Kaper J.B. (1998) Diarrheagenic Escherichia coli. Clinical Microbiology Re- views, 11: 142-201.
  • Patel J.R., Bhagwat A.A., Sanglay G.C. and Solo- mon M.B. (2006) Rapid detection of Salmonella from hydrodinamic pressure-treated poultry us- ing Molecular Beacon Real-Time PCR. Food Microbiology, 23: 39-46.
  • Perelle S., Dilasser F., Grout J. and Fach P. (2004) Detection by 5’-nuclease PCR of shiga-toxin producing Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157:H7, as- sociated with the world’s most frequent clini- cal cases. Molecular and Cellular Probes, 18: 185-192.
  • Rosmini M.R., Signorini M.L., Schneider R. and Bonazza J.C. (2004) Evaluation of two alterna- tive techniques for counting mesophilic aerobic bacteria in raw milk. Food Control 15: 39-44.
  • Schönenbrücher V., Mallinson E.T. and Bülte M. (2007) A comparison of standard cultural meth- ods for the detection of foodborne Salmonella species including three new Chromogenic Plat- ing Media. International Journal of Food Mi- crobiology, 123: 1-29.
  • Takahashi H., Kimura B., Tanaka Y., Shinozaki J., Suda T. and Fujii T. (2009) Real-Time PCR and enrichment culture for sensitive detection and enumeration of Escherichia coli. Journal of Microbiological Methods, 79: 124-127.
  • Temelli S., Dokuzlu C. and Şen C.K.M. (2004) Determination of microbiological contamina- tion sources during frozen snail meat processing stages. Food Control, 17: 22-29.
  • Ulukanlı Z., Çavlı P. and Tuzcu M. (2006) Detection of Escherichia coli O157:H7 from beef Doner Kebabs sold in Kars. Gazi University Journal of Science 19: 99-104.
  • Ünlütürk A. and Turantaş F. (2003) Gıda Mikro- biyolojisi. Meta Basım Matbaacılık Hizmetleri. İzmir 3. Basım Meta Basım Matbaacılık Hizmetleri pp. 90-91.
  • Yıldırım I., Uzunlu S. and Topuz A. (2005) Effect of gamma irridiation on some principle microbio- logical and chemical quality parameters of raw Turkish meat ball. Food Control, 16: 363-367.

Türkiye’de baharatlarda Escherichia coli O157:H7’nin belirlenmesinde real-time PCR ve konvansiyonel kültürel yöntemin karşılaştırılması

Year 2012, Volume: 71 Issue: 2, 113 - 120, 25.12.2012

Abstract

Bu çalışmada, baharatlarda E. coli O157:H7’nin belirlenmesinde Real-Time PCR ve kültürel yöntemlerinin karşılaştırılması için 66 farklı baharat örneği analiz edilmiştir. Bunun için 66 farklı baharat örneği (ham madde) İzmir’in çeşitli yerlerinden temin edilmiştir. Örneklerdeki toplam mikrobiyal yükün belirlenmesi için E. coli O157:H7 yanında toplam aerobik mezofilik bakteri, koliform, fekal koliform sayımları yapılmıştır ve Salmonella sp. varlığına bakılmıştır. Bütün örneklerde ortalama değerler aerobik mezofilik bakteri sayımı 5.1x103 -2.0x108 CFU/g ve koliform sayımı 8.0 x 102 CFU/g olarak bulunmuştur. Hiçbir örnekte Salmonella sp. bulunmamıştır. Kültürel yöntemle bütün örnekler E. coli O157:H7 bakımından negatif bulunurken Real-Time PCR yöntemiyle 4 tane örnek pozitif bulunmuştur. Bu çalışmada RealTime PCR yönteminin kültürel yönteme göre daha güvenilir olduğu gözlenmiştir. Bunun yanında kültürel yöntemle sonuç 4 günde alınırken Real-Time PCR yöntemiyle sadece 20 saatte sonuç alınmıştır

References

  • Akkaya L. and Alişarlı M. (2006) Afyonkarahisar’da tüketime sunulan peynirlerde Listeria mono- cytogenes ve Salmonella spp. varlığının be- lirlenmesi. Yüzüncü Yıl Üniversitesi Veteriner Fakültesi Dergisi, 17: 87-91.
  • Alişarlı M. and Akman HN. (2004) Perakende satılan kıymaların Escherichia coli O157:H7 yönünden incelenmesi. Yüzüncü Yıl Üniversitesi Veteriner Fakültesi Dergisi, 15: 65-69.
  • Ateş M., Özkızılcık A. and Tabakoğlu C. (2011) Microbiological Analysis of Stuffed Mussels Sold in The Streets. Indian Journal of Micro- biology, 51: 350-354.
  • Bhagwat A.A. (2002) Simultaneous detection of Escherichia coli O157:H7, Listeria monocyto- genes and Salmonella strains by real-time PCR. International Journal of Food Microbiology, 84: 217-224.
  • Catarame T.M.G., O’hanlon K.A., McDowell D.A., Blair I.S. and Duffy G. (2006) Comparison of a Real-Time Polymerase Reaction Assay with a cultural method for the detection of Salmonella in retail meat samples. Journal of Food Safety, 26: 1-15.
  • Chigbu P., Gordon S. and Strange R.T. (2005) Fe- cal coliform bacteria disapperance rates in a North-central Gulf of Mexico estuary. Estuarine, Coastal and Shelf Science, 65: 309-318.
  • Chui S. (2004) Detection and characterization of Esherichia coli O157:H7 and Salmonella in food, PhD, University of Maryland pp. 3.
  • Ellingson E.L.J., Koziczkowski J.J., Anderson L.J., Carlson A.S. and Sharma K.V. (2005) Rapid PCR detection of enterohemorrhagic Esch- erichia coli (EHEC) in bovine food products and feces. Molecular Cell Probes, 19: 213-217.
  • Espy M.J., Uhl J.R., Sloan L.M., Buckwalter S.P., Jones M.F., Vetter E.A., Yao J.D.C., Wengenack N.L. and Rosenblatt J.E. (2006) Real-Time PCR in clinical microbiology: Applications for rou- tine laboratory testing. Clinical Microbiology Reviews, 19: 165-256.
  • Feng P., Weagant S.D., Grant M.A. and Burkhardt W. (2002) BAM: Enumeration of Escherichia coli and the Coliform Bacteria. Bacteriologi- cal Analytical Manual 8th Edition, Revision A, Chapter 4.
  • Fitzmaurice J. (2008) Detection of verotoxin genes VT 1 and VT 2 in Escherichia coli O157:H7 in minced beef using Immunocapture and Real- Time PCR. In: Louise O’Connor (Ed.) Diag- nostic Bacteriology Protocols, New Zealand 345: 91-96.
  • Geissler K., Manafi M., Amoros I. and Alonso J.L. (2000) Quantitative determination of total co- liforms and Escherichia coli in marine waters with chromogenic and fluorogenic media. Jour- nal of Applied Microbiology 88: 280-285.
  • Islam A.M., Heuvelink A.E., Talıkder A.K. and Boer E.D. (2006) Immunoconcentration of shiga toxin-producing Escherichia coli O157 from animal faeces and raw meats by using dyna- beads anti-E. coli O157 and the VIDAS System. International Journal of Food Microbiology, 109: 151-156.
  • Kök F., Özbey G. and Muz A. (2007) Aydın İlinde Satışa Sunulan Fermente Sucukların Mikro- biyolojik Kalitelerinin İncelenmesi. Fırat Üni- versitesi Sağlık Bilimleri Dergisi, 21: 249-252.
  • Liu Y., Gilchrist A., Zhang J., and Li XF. (2008). Detection of Viable but Nonculturable Esch- erichia coli O157:H7 Bacteria in Drinking Water and River Water. Applied and Environmental Microbiology 74: 1502-1507.
  • Nataro J.P. and Kaper J.B. (1998) Diarrheagenic Escherichia coli. Clinical Microbiology Re- views, 11: 142-201.
  • Patel J.R., Bhagwat A.A., Sanglay G.C. and Solo- mon M.B. (2006) Rapid detection of Salmonella from hydrodinamic pressure-treated poultry us- ing Molecular Beacon Real-Time PCR. Food Microbiology, 23: 39-46.
  • Perelle S., Dilasser F., Grout J. and Fach P. (2004) Detection by 5’-nuclease PCR of shiga-toxin producing Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157:H7, as- sociated with the world’s most frequent clini- cal cases. Molecular and Cellular Probes, 18: 185-192.
  • Rosmini M.R., Signorini M.L., Schneider R. and Bonazza J.C. (2004) Evaluation of two alterna- tive techniques for counting mesophilic aerobic bacteria in raw milk. Food Control 15: 39-44.
  • Schönenbrücher V., Mallinson E.T. and Bülte M. (2007) A comparison of standard cultural meth- ods for the detection of foodborne Salmonella species including three new Chromogenic Plat- ing Media. International Journal of Food Mi- crobiology, 123: 1-29.
  • Takahashi H., Kimura B., Tanaka Y., Shinozaki J., Suda T. and Fujii T. (2009) Real-Time PCR and enrichment culture for sensitive detection and enumeration of Escherichia coli. Journal of Microbiological Methods, 79: 124-127.
  • Temelli S., Dokuzlu C. and Şen C.K.M. (2004) Determination of microbiological contamina- tion sources during frozen snail meat processing stages. Food Control, 17: 22-29.
  • Ulukanlı Z., Çavlı P. and Tuzcu M. (2006) Detection of Escherichia coli O157:H7 from beef Doner Kebabs sold in Kars. Gazi University Journal of Science 19: 99-104.
  • Ünlütürk A. and Turantaş F. (2003) Gıda Mikro- biyolojisi. Meta Basım Matbaacılık Hizmetleri. İzmir 3. Basım Meta Basım Matbaacılık Hizmetleri pp. 90-91.
  • Yıldırım I., Uzunlu S. and Topuz A. (2005) Effect of gamma irridiation on some principle microbio- logical and chemical quality parameters of raw Turkish meat ball. Food Control, 16: 363-367.
There are 25 citations in total.

Details

Primary Language English
Journal Section Makaleler
Authors

Asli Ozkizilcik

Mustafa Ates This is me

Burcu Cerci This is me

Publication Date December 25, 2012
Submission Date December 25, 2012
Published in Issue Year 2012 Volume: 71 Issue: 2

Cite

AMA Ozkizilcik A, Ates M, Cerci B. Comparison of real-time PCR and conventional cultural methods to detect Escherichia coli O157:H7 in spices in Turkey. Eur J Biol. November 2012;71(2):113-120.