abbas morovvati

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  • The diarrhea and other symptoms of EPEC infections probably are caused by bacterial invasion of host cells and interference with normal cellular signal transduction, rather than by production of toxins.
  • Studies with adult volunteers have demonstratedthat intimin, pEAF and BFP are essential virulence deter-minants of EPEC [10-12]. Interestingly, there is evidencethat a subset of EPEC strains, known as atypical EPEC(aEPEC), which lack pEAF and BFP, are also pathogenic
  • EPEC strains may be either typical, producing bundle forming pili(encoded by the bfpA gene) that mediatelocalised adherence on cultured epithelial cells, or atypical, which are negative for these appendages but show localised like adherence,diffuse adherence or aggregative adherence patterns and produce diarrhae in developing worlds.Virulance gene is bfp for typical and non typical
  • abbas morovvati

    1. 1. 2
    2. 2. IntroductionEnteric bacterial diseasesEtiologic bacteriological diagnosis by classical cultures techniques do not allow detection and identification of the most frequent diarrheagenic Escherichia coli in our countries as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC).
    3. 3. EPEC is a leading cause of diarrhea in developing countries.In industrialized countries, the frequency of these organisms has decreased, but they continue to be an important cause of diarrhea .
    4. 4. EPEC was first reported by John Bray in 1945as the cause of diarrhea outbreak in London 5
    5. 5. Classification of Enteropathogenic E. coliPathotype Clin Features Epidem. Features Virulence factorsEPEC Watery diarr., Infants, Bundle forming pilus, vomiting Developing attaching-effacing countriesEHEC Watery diarr., Food & water Shiga toxins, attaching- Hg. colitis borne effacingETEC Watery diarr Childhood diarr., Pili, ST & LT entero Travelers diarr. toxinsEAEC Diarr with Childhood diarr. Pili, cytotoxins mucusEIEC Dysentery/ Food borne Cellular invasion, intra watery diarr cellular motility
    6. 6. Epidemiology
    7. 7. Laboratory diagnosis 1- Molecular methodes 2- immunological 9
    8. 8. Material and methods Gel bfpExtraction PCR Design Cloning Sequencing
    9. 9. Primer Accesion number :FM180569 Cinnagen 11
    10. 10. Primer Design Primer Name5’-GGA AGT CAA ATT CAT GGG GGT AT bfp F bfp R5’-GGA ATC AGA CGC AGA CTG GTA GT
    11. 11. PCR and Gradient PCR Temperature Time Cycle(s)Gradient PCR 94 5 min 1 for samples g=5 94 45 sec 52 1min 40 72 1min 72 5 min 1 Primer F 1 l Primer R 1 l10mM dNTP Mix10X Taq 22 l buffer Containing 1. 5mm MgCl2 Taq Polymerase 1U DNA 1 l
    12. 12. Gel agarose electrophoresis 1 2 3 4 5 6 7 8 PCR Product= 250 bp 1= ladder 100bp plus fermentas 2-7 = sample 8 = negative control 250 bp
    13. 13. Gel Extraction This methode did by Commercial Corebio Kit
    14. 14. Cloning Strain:JM107 Vector:pTZ57R/T
    15. 15. Selection and Matrix
    16. 16. Colony PCR 1 2 3 4 5 6 7 8 9 10 111= ladder 100 bp2-11= white colonyhad insert12 = blue colonySamples 3 , 5, 7selected for cultured inplasmid
    17. 17. Plasmid extraction Plasmid extract by bioneer comersial kit and then PCR for confirm our extraction
    18. 18. Digestion 1 2 3 4 Digestion with BamHI and XbaI Enzyme 1X Tango insert= 250 bp 1= ladder 100bp plus fermentas 2 =un digest 3 = digest 4 = ladder 1kb 250 bp
    19. 19. SEQUENCING
    20. 20. 24
    21. 21. 25

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