1. COLLEGE OF VETERINARY MEDICINE
HARAMAYA UNIVERSITY
COLLEGE OF VETERINARY MEDICINE
SENIOR SEMINAR PRESENTATION ON THE TITLE
MOLECULAR TECHNIQUES APPLICATION FOR E.COLI O157:H7 DETECTION
SET BY NATENEAL TAMERAT
APRIL 17/2013

2. Introduction Food born pathogen threat the fast growing
market of food and revealed a hazard for the
Public health.
One of them is E. coli serotype O157:h7 and can result to haemorrhagic colitis, heamolytic ureamic syndrome and thrombotic thrombocytopenic purpura.
So Rapid and reliable molecular Techniques are mandatory for detection of this pathogen...

3. History and genomics of E. coli
“Once we understanding the biology of e .coli we will understand the biology of elephant.” “Jacque Monad”” “
History and genomics of E. coli
the German bacteriologist Theodor Escherich first identify E. coli in 1885.
But it was in 1982 the E. coli serotype O157:H7 implicated for out break of heamorrhagic colitis and heamolytic uraemic syndrome.

4. E. coli chromosomes range from 4.500 to 5.520
million base pairs and the E. coli O157:H7 is closer to the upper limit.
It has large plasmid and pathogenicity island which contain different gene responsible for the virulence of this serotype .
There are Two assumption about the evolutionary origin of O157:H7, one lineage origin and two lineage origin.

5. Advantages of molecular technique over conventional methods.
Time saving
Reliable
Efficient
Simple

6. Molecular techniques for detection of O157:H7
PCR Variant
Emerging technology
Genomic tehniques
Microarray
PFGE
M- PCR
RFLP
Biosenser
R-PCR
Taq man
RT-PCR

7. Pulse field gel electrophorosis

8. Restriction Fragment Length polymorphism

9. PCR
It is a device which amplify the template DNA exponentially to detect O157:H7 from the sample.

10. Phases of PCR

11. VARIANT OF PCR Multiplex PCR
Variant of PCR which allows several targets of O157:H7 to be co-amplified simultaneously in the same reaction by combining several primer pairs.
REAL TIME PCR
detect the presence of target gene of O157:H7 at real time.
REVERSE TRANSCRIPTASE PCR
Detect Target RNA of viable O157:H7.

12. R-PCR

13. Reverse Transcriptase PCR

14. Microarray
Microarray is a device that allows thousands specific DNA or RNA sequences of O157:H7 to be detected simultaneously on a small slide.
. For these applications, labeled nucleic acid
targets are hybridized to a microarray chip where upon target probe duplexes are typically detected using some type of fluorescent signal system.

15. MICROARRAY

16. Different molecular techniques with their merit and demerit.
Advantage
Disadvantage
PFGE
Good discrimination
Reproducible and standardize
Require high Technical skill
RFLP
Partial genome technique
Difficult to digitalize
and standardize
M-PCR
Detection of multiple pathogenic
gene
Post PCR analysis
R-PCR
Save time and labor
Quantify the product
Single gene base
RT-PCR
Detect viable pathogen
MICROARRAY
PCR independent
Fast
Expensive
Knowledge and Training

17. CONCLUSION
Although conventional procedures remain an integral part of detection methods they are laborious and time consuming. But molecular identification of virulence genes has greatly facilitated development of detection and genotyping of O157:H7.
Since each technique have its own merit and demerit, the decision for the selection of detection technique will involve striking a balance between several factors according the existing situation.

18. RECOMMENDATIONS
based on the above conclusion, the following recommendations are forwarded:
Isolation of O157:H7 followed by molecular detection method like m-PCR is considered as the method of choice in ideal condition.
In Ethiopia E. coli O157:H7 associated disease status and distribution should be assessed.
Technical and economical support should be given for our laboratory in molecular techniques.