Volume 20, Issue 3 (Iranian South Medical Journal 2017)                   Iran South Med J 2017, 20(3): 267-277 | Back to browse issues page

XML Persian Abstract Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
Bakhshi M, Ebrahimi F, Nazarian S, Tarverdizade Y, Sadeghi D. Identification of E. coli O157:H7 by Using Specific Primers for rfbE and stx2b Genes. Iran South Med J 2017; 20 (3) :267-277
URL: http://ismj.bpums.ac.ir/article-1-878-en.html
Identification of E. coli O157:H7 by Using Specific Primers for rfbE and stx2b Genes
Mostafa Bakhshi1 , Firouz Ebrahimi1 , Shahram Nazarian * 2, Yousef Tarverdizade1 , Davood Sadeghi1
1- Department of Biology, School of Basic Science, Imam Hossein University, Tehran, Iran
2- Department of Biology, School of Basic Science, Imam Hossein University, Tehran, Iran , kpnazari@ihu.ac.ir
Abstract:   (8951 Views)

Background: E. coli O157:H7 is one of the intestinal pathogens which can cause severe lesions in the gastrointestinal system. These bacteria can produce toxins and are the main cause of hospital infections. Their detection is usually done by culture on sorbitol-MacConkey agar which is a time-consuming test. The aim of this study was to develop a rapid, yet accurate method to identify this bacterium using a PCR based technique.
Material and Methods: rfbE and stx2b genes were selected for identification of specific E. coli O157:H7 strain. Then amplification was performed by PCR following designing specific primers. Sorbitol-MacConkey agar was used to verification of growth ability of selected colonies during PCR.
Results: By the appearance of the bonds belong to rfbE and stx2B genes on an agarose gel, the ability of designed primers for gene detection in samples of E .coli O157:H7 was verified. A sorbitol-MacConkey agar medium was used to evaluate the growing potency of colonies selected during PCR.
Conclusion: In this study we demonstrated that we could develop a fast, accurate, and relatively comfortable method for detection of E. coli O157:H7 strain by using PCR technique and specific primers instead of using current methods.
 
 

Keywords: Identification of E. coli O157:H7 strain, polymerase chain reaction, rfbE gene, stx2b gene
Full-Text [PDF 650 kb]   (2978 Downloads)    
Type of Study: Original | Subject: Biochemistry. Cell Biology and Genetics
Received: 2016/11/7 | Accepted: 2017/01/9 | Published: 2017/07/5
References
1. Lim JY, Yoon J, Hovde CJ. A brief overview of Escherichia coli O157:H7 and Its plasmid O157. J Microbiol Biotechnol 2010; 20(1): 5-14.
2. Tarr PI, Schoening LM, Yea YL, et al. Acquisition of the rfb-gnd Cluster in Evolution of Escherichia coli O55 and O157. J Bacteriol 2000; 182(21): 6183-91. [DOI:10.1128/JB.182.21.6183-6191.2000]
3. Maurer JJ, Schmidt D, Petrosko P, et al. Development of Primers to O-antigen biosynthesis genes for specific detection of Escherichia coli O157 by PCR. Appl Environ Microbiol 1999; 65(7): 2954-60.
4. Gyles CL. Shiga toxin-producing Escherichia coli: an overview. J Anim Sci 2007; 85(13 Suppl): E45-62. [DOI:10.2527/jas.2006-508]
5. Law D. Virulence factors of Escherichia coli O157 and other Shiga toxin-producing E. coli. J Appl Microbiol 2000; 88(5): 729-45. [DOI:10.1046/j.1365-2672.2000.01031.x]
6. Guth B, Prado V, Rivas M. Shiga Toxin Producing Escherichia coli. In: Torres AG, ed. Pathogenic Escherichia coli in Latin America. Sharjah: Bentham, Science Publishers, 2010, 65-83.
7. Ojeda A, Prado V, Martinez J, et al. Sorbitol-negative phenotype Enterohemorrhagic Escherichia coli strains of different serotypes and from different sources. J Clin Microbiol 1995; 33(8): 2199-201.
8. Noveir MR, Dogan HB, Kadir Halkman A. A note on Escherichia coli O157:H7 serotype in Turkish meat products. Meat Sci 2000; 56(4): 331-5. [DOI:10.1016/S0309-1740(00)00058-9]
9. Saeed AY, Ibrahim KS. Identification of Escherichia coli O157 in sheep and goats using PCR technique. IOSR-JAVS 2013; 6(2): 30-2. [DOI:10.9790/2380-0623032]
10. Tamerat N, Muktar Y, Shiferaw D. Application of molecular diagnostic techniques for the detection of E. coli O157:H7: a review. J Vet Sci Technol 2016; 7(362): 1-9. [DOI:10.4172/2157-7579.1000362]
11. Puttalingamma V, Niveditha S. A review: detection of Escherichia coli O157:H7. Int J Pharm Pharm Sci Res 2014; 4(3): 49-52.
12. Farajzadeh Sheikh A, Rostami S, Amin M, et al. Isolation and identification of Escherichia coli O157:H7 from ground beef hamburgers in Khuzestan Province, Iran. Afr J Microbiol Res 2013; 7(5): 413-7. [DOI:10.5897/AJMR12.2239]
13. Rychlik W. OLIGO 7 Primer Analysis Software. In: Yuryev A, editors. PCR Primer Design, Totowa: Humana Press, 2007, 35-59. [DOI:10.1007/978-1-59745-528-2_2]
14. Nazarian S, Mousavi Gargari SL, Rasooli I, et al. Prevalent phenotypic and genotypic profile of enterotoxigenic Escherichia coli among Iranian children. Jpn J Infect Dis 2014; 67(2): 78-85. [DOI:10.7883/yoken.67.78]
15. Mousavi SL, Rasooli I, Nazarian S, et al. Simultaneous detection of Escherichia coli O157:H7, toxigenic Vibrio cholerae, and Salmonella typhimurium by multiplex PCR. Arch Clin Infect Dis 2009; 4(2): 97-103.
16. Mesapogu S, Jillepalli CM, Arora DK. Agarose Gel Electrophoresis and Polyacrylamide Gel Electrophoresis: Methods and Principles. In: Arora DK, Das S, Sukumar M, editors. Analyzing microbes: Manual of molecular biology techniques. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013, 73-91. [DOI:10.1007/978-3-642-34410-7_5]
17. Tahamtan YE, Puorbakhsh SA, Shekarforoush SS. PCR detection of Escherichi coli O157:H7 directed from slaughtered cattle in Shiraz, Iran. Arch Razi Ins 2006; 61(1):1-6.
18. Tavakoli HR, Ghorban Alizadegan M, Najafi A, et al. Detection of toxicogenic E.coli 0157:H7 by PCR. Iran J Infect Dis Trop Med 2010; 15(49): 49-53. (Persian) Adeli Z, Firoozeh F, Zibaei M, et al. Prevalence of Shiga toxin and Intimine genes in Shiga toxin-producing Escherichia coli isolated from urine samples in Lorestan, Iran. Feyz 2013; 17(2): 188-94. (Persian) Kargar M, Daneshvar M, Homayoun M. Surveillance of Virulence Markers and Antibiotic Resistance of Shiga toxin Producing E.coli O157:H7 Strains from Meats Purchase in Shiraz. Iran South Med J 2011; 14 (2) :76-83 (Persian)
19. Hu Y, Zhang Q, Meitzler JC. Rapid and sensitive detection of Escherichia coli O157:H7 in bovine faeces by a multiplex PCR. J Appl Microbiol 1999; 87(6): 867-76. [DOI:10.1046/j.1365-2672.1999.00938.x]
20. Fode-Vaughan KA, Maki JS, Benson JA, et al. Direct PCR detection of Escherichia coli O157: H7. Lett Appl Microbiol 2003; 37(3): 239-43. [DOI:10.1046/j.1472-765X.2003.01386.x]
21. Campbell GR, Prosser J, Glover A, et al. Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR. J Appl Microbiol 2001; 91(6): 1004-10. [DOI:10.1046/j.1365-2672.2001.01465.x]
22. Fratamico PM, Strobaugh TP. Simultaneous detection of Salmonella spp and Escherichia coli O157:H7 by multiplex PCR. J Ind Microbiol Biot 1998; 21(3): 92-8. [DOI:10.1038/sj.jim.2900520]
23. Matise I, Shelton M, Phillips G, et al. Sensitive PCR Method for Detection of Escherichia coli 0157:H7 and Other Shiga toxin-producing Bacteria in Ground Meat. Swine Res Rep 1997; 33: 1-6.
24. Ayaz ND, Copuroglu G, Ormeci E, et al. Presence of Staphylococcus aureus and Shiga Toxigenic Escherichia coli O157:H7 in Raw Meat in Agri, Turkey Int J Enteric Pathog 2016; 29: 1-7.
25. Fortin NY, Mulchandani A, Chen W. Use of Real-Time Polymerase Chain Reaction and Molecular Beacons for the Detection of Escherichia coli O157:H7. Anal Biochem 2001; 289: 281-288. [DOI:10.1006/abio.2000.4935]
26. Khatami F, Heidari M, Khatami M. Rapid Detection of Escherichia coli O157: H7 by Fluorescent Amplification–Based Specific Hybridization (FLASH) PCR. Iran Red Crescent Med J 2012; 14(9): 594-8.
27. Atabakhsh P, Amin MM, Mortazavi H, et al. Identification of Total and Fecal Coliforms and Heterotrophic to Microbiological Method and E. coli O157:H7 to Immunological, and Real Time PCR Methods in Isfahan Water Treatment Plant. Iran J Health & Environ 2010; 3(3): 335-46. (Persian)
28. Almeida C. Sousa JM, Rocha R, et al. Detection of Escherichia coli O157 by Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) and Comparison to a Standard Culture Method. Appl Environ Microb 2013; 79(20): 6293-6300. [DOI:10.1128/AEM.01009-13]
Send email to the article author

Rights and Permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

© 2024 CC BY-NC 4.0 | Iranian South Medical Journal

Designed & Developed by: Yektaweb