molecular techniques application for e.coli o157:h7 detection
TRANSCRIPT
HARAMAYA UNIVERSITY
COLLEGE OF VETERINARY MEDICINE
SENIOR SEMINAR PRESENTATION MOLECULAR TECHNIQUES APPLICATION FOR E.COLI
O157:H7 DETECTION
Set By Nateneal Tamerat
APRIL 17/2013
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...
“Once we understanding the biology of e .coli we will understand the biology of elephant.”
“Jacque Monad””“
History and genomics of E. colithe 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.
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.
Advantages of molecular technique over conventional methods.
.Time saving ReliableEfficient Simple
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
Pulse field gel electrophorosis
Restriction Fragment Length
polymorphism
PCR
It is a device which amplify the template DNA exponentially to detect O157:H7 from the sample.
Phases of PCR
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.
R-PCR
Reverse Transcriptase PCR
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.
MICROARRAY
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 digitalizeand standardize
M-PCR Detection of multiple pathogenicgene
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
ExpensiveKnowledge and Training
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.
RECOMMENDATIONSbased 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.