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

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...

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.

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 Reliable Efficient Simple

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

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. 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

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.

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.