Biotraceability in Food and Feed Chains--Course Descriptions

A technical workshop on molecular biology methods used, or possible to use, in biotraceability

Overview
The workshop is intended to give some level of understanding of microbiology to those not familiar with the discipline. It will bring the various disciplines of BIOTRACER closer together and foster an understanding of microbiology among non-microbiologists, one of the aims of BIOTRACER. The aims of the workshop are to place these technologies in context and to give some basic level of understanding of the technologies to non-specialists. The workshop will be presented by people working with thes technologies who will give a 30 min presentation on the technology, explaining it in a simple way.

Methods covered will include polymerase chain reaction (PCR), pulse field gel electrophoresis (PFGE), genomotyping and microarrays.

PCR
DNA/RNA amplification and quantification in areas such as diagnostic PCR and molecular biology has been greatly improved by the introduction of real-time PCR technology. While this technology has tremendous potential for accurate and sensitive quantification, careful considerations regarding experimental design, sample preparation and reagent optimisation are required before it can be used correctly.

PFGE
PFGE is the comparison of large genomic DNA fragments after digestion with a restriction enzyme. The basic concept of interpretation of this experiment is the following: if one is comparing two strains that are clonal (i.e. the same strain), the sites at which the restriction enzymes act on the DNA and the length between these sites would be identical. Therefore, after digestion of the DNA and electrophoresis through an agarose gel, if the DNA banding patterns between any two isolates is identical, then these isolates are considered the same strain. Conversely, if two isolates are not the same strain, then the sites at which the restriction enzymes act on the DNA and the length between these sites would be different; thus their DNA banding patterns will be different and the strains are considered different.

Microarrays
In general you can think of a microarray as a grid of DNA spots. Each spot has a unique DNA sequence, different from the DNA sequence of the other spots around it. Thus each spot will hybridize only to its complementary DNA strand. One thing that makes microarrays so exciting is the number of DNA probes that it is possible to place on a microarray. Already there are microarrays with probes for every gene in a bacterium, and others with over 19,000 human DNA spots. This allows researchers to observe the response of whole genomes to various stimuli instead of one gene at a time. Genome-composition analysis using microarrays, or 'genomotyping', can be used to categorize genes into 'present' and 'divergent' categories based on the level of hybridization signal, and thus compare genomes.

Outline
Introduction                                                        Jenny Schelin
Animals as sources of food pathogens                   Martin Wagner
Conventional detection methods in microbiology     To be decided
PCR, qPCR                                                          Peter Radstorm

Break

PFGE                                                                   Kieran Jordan
Microarrays/Genomotyping                                   Richard Stabler
Discussion/Conclusion

Pre-workshop Required Reading*
http://en.wikipedia.org/wiki/Molecular_microbiology#Technology
http://en.wikipedia.org/wiki/Food_microbiology#Foodborne_pathogens
*Required for PhD students.


Post-workshop Reading Material
1. Löfström C, Knutsson R, Engdahl Axelsson C, Rådström P (2004) Rapid and specific detection of Salmonella spp. in animal feed samples by PCR after culture enrichment. Appl Environ Microbiol 70:69-75

2. Lübeck PS, Wolffs P, On SL, Ahrens P, Rådström P, Hoorfar J (2003) Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: assay development and analytical validation. Appl Environ Microbiol 69:5664-5669

3. Call D.R. 2005. Challenges and opportunities for pathogen detection using DNA microarrays.
Crit. Rev. Microbiol 31: 91-99

4. Nick Dorrell, Stewart J Hinchliffe and Brendan W Wren. 2005 Comparative phylogenomics of pathogenic bacteria by microarray analysis. Current opinion in microbiology 8: 620-626.