Molecular Techniques Application for E.Coli O157:H7 Detection

1. COLLEGE OF VETERINARY
MEDICINE
SENIOR SEMINAR PRESENTATION
MOLECULARTECHNIQUES APPLICATIONFOR 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. “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
Genomic
tehniques
PCR Variant
Emerging
technology
PFGE
RFLP
M- PCR
R-PCR
RT-PCR
Microarray
Taq
man
Biosenser

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.
Techniques 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 Single gene base
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.