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Conventional vs Capillary Electrophoresis of RFLP Fragments and PCR Products for Sensitive and Rapid Subtyping of Salm - OZ0311

Description
This proposal directly addresses para 119B of the MAFF animal health and welfare requirements document 1999/2000, to develop sensitive and specific molecular subtyping techniques to enable accurate studies of the epidemiology of Salmonella species.

The objective of this proposal is to compare the relative adavantages and disadvantages of different electrophoretic techniques for molecular subtyping of Salmonella. Thus, Amplified Restriction Fragment Length Polymorphism (AFLP), Pulsed-field Gel Electrophoresis (PFGE), Random Amplification of Polymorphic DNA (RAPD), Restriction Fragment Length Polymorphism (RFLP) and ERIC-PCR will be investigated for their ability to discriminate between serovars and within serovars and lysovars of Salmonella obtained from veterinary, human and food sources, as a standard against which Capillary Gel Electrophoresis can be compared for the purposes of this project.

The use of capillary electrophoresis along with rapid DNA preparation techniques will be developed and adapted to replace the slower electrophoretic procedures currently used in these techniques as a way of increasing the discrimination, rapidity and potential automation of the typing process in all except ribotyping and PFGE which is not amenable to linear electric field separation.

The results of this work will lead to more rapid and efficient use of molecular subtyping techniques in ecological and epidemiological studies and encourage standardisation of methodology by:

• Forming a basis on which Salmonella typing schemes may be continuously improved
• Reducing time and reducing their cost
• Producing multiple information concerning a strain in a single separation
• Improving information flow
• Making decisions more sure and more rapid
• Helping devise better and more effective HACCP schemes at all points in the food chain
• Contribute positively to the control of the Salmonella hazard at all points in the food chain from farm to fork.
• Form a basis for a generic approach to the rapid typing of all microorganisms of concern in animals, humans
and foods
Objective
1. Review the state of the science concerning molecular typing techniques and their application for typing of microorganisms in animals, humans and foods with special emphasis on Salmonella and with reference to other food –borne zoonoses.

2. For the most commonly isolated serovars of Salmonella: compare at least 20 strains each of the most commonly isolated serovars of Salmonella from animals and foods (S. enteritidis, S. hadar, S. typhimurium and S. virchow) using AFLP, PFGE, RAPD, REP/ERIC-PCR and RFLP. At least 2 strains of each phage type where this is possible of S. enteritidis and S. typhimurium will be studied. PHLS will be responsible for PFGE and RAPD testing and CSL the rest.

3. For rarer serovars: compare at least 10 strains each of S. derby and S. montevideo and 2 strains (a type strain and a clinical isolate) of 100 others using the 6 techniques in 2. Rarer serovars will be chosen on their importance in animals, food and humans.

4. Develop capillary electrophoresis protocols for separation of RFLP fragments and PCR products from, AFLP, RAPD, RFLP, and REP/ERIC-PCR. Capillary gel electrophoresis will be carried out at CSL.

5. Apply capillary electrophoresis to typing of strains and compare results.
Project Documents
• Final Report : Conventional vs capillary electrophoresis of RFLP fragments and PCR products for sensitive, rapid subtyping of Salmonella   (591k)
Time-Scale and Cost
From: 1999

To: 2004

Cost: £373,347
Contractor / Funded Organisations
Central Science Laboratory
Keywords
Animal Health              
Biotechnology              
Control              
Diagnosis              
Plants and Animals              
Salmonella              
Zoonoses              
Fields of Study
Animal Health