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Development of a novel molecular typing method for comparison of food borne pathogens, using VTEC as a model organism
Project Code: B01014
The overall objective of the work was to carry out molecular analyses to identify polymorphic DNA markers that reproducibly represent specific traits in foodborne VTEC, to then establish and assess the framework of a working database to catalogue both DNA markers and known phenotypic characteristics.
During the course of the study a catalogue of 400 Scottish O157 isolates from a variety of sources was produced, consisting of representative isolates from a variety of biological and environmental sources. In addition, a robust protocol for the generation of AFLP profiles was developed, based on the method provided by PE Applied Biosystems with their commercially available Microbial Fingerprinting kit. The method was evaluated in a two-centre study that demonstrated the reproducibility and accuracy of data produced by the AFLP technique. In particular, the use of the ABI377 sequencer and inclusion of internal size markers ensures highly accurate fragment sizing, allowing meaningful comparison of fingerprints to be performed. To ensure reproducible data interpretation and comparability of results between analysts and laboratories, detailed criteria for profile acceptance were formulated. In addition, methods for normalisation of data were developed to ensure uniformity of profile peaks between runs.
Evaluation of many commercially available PCR primer and restriction enzyme combinations, and several novel enzyme/primer combinations, has been performed to maximise the discriminatory power of the technique. However, despite extensive investigation, no primer and enzyme combination was found that effectively distinguished the majority of the Scottish O157 isolates, making the method of little practical use for the identification of these samples. Lack of discrimination prevented both correlation of specific profile markers with known phenotypic or genotypic traits, and association of particular strain types with their environmental origin. The poor level of isolate discrimination obtained did not permit more insightful epidemiological analysis of the distribution of isolates, or analysis of potential reservoirs of human infection.
The format of the fAFLP data is accessible to database storage and automated searching, facilitating comparison of information between laboratories. A database has been constructed to allow both storage of isolate profiles and comparison of molecular and phenotypic characteristics of strains, using Microsoft Access. The database facilitates matching of AFLP profiles of unknown samples with those already entered into the database, and can also be used to match isolates using other known characteristics.
In conclusion, fAFLP confers several advantages over currently used typing methods, including the reproducibility and accuracy of the technique, and the accessibility of the information to database storage. However, the lack of sensitivity of the method and poor discrimination obtained in this study makes AFLP profiling unsuitable for comparison of the Scottish O157 isolates.
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