Review of the Food-borne Zoonoses Research ... - ARCHIVE: Defra

To test the suitability of the AFLP method for sensitive characterisation of Salmonella
species, over 400 isolates were selected for investigation, from the collection of the
Scottish Salmonella Reference Laboratory at Stobhill Hospital in Glasgow. A catalogue of
representative strains was produced, including 243 isolates of human origin, 83 animal
isolates, 37 from a variety of foods and 26 strains acquired from environmental sources.
The isolates had already been characterised by classical typing methods by the Scottish
Reference Laboratory. In addition, the majority of the strains had been typed using
PFGE, and their antibiotic resistance profile determined. DNA extracts from the
resuscitated Salmonella strains were provided by the Scottish Salmonella Reference
Laboratory for AFLP analysis at LGC.
The first stage of the project focused on development of a robust protocol for the
generation of AFLP profiles. To facilitate inter-laboratory comparison it was decided to
utilise commercially available AFLP reagents provided as a Microbial Fingerprinting Kit.
However, in the last year of the project, the highly variable quality of commercially
available reagents necessitated a change to independently-sourced materials. The effect
of a variety of parameters on the robustness and quality of AFLP profiles was assessed,
and an optimised protocol was developed. To promote reproducible data interpretation
and comparability of results between analysts and laboratories, methods for
normalisation of data and criteria for AFLP profile acceptance were developed. However,
the inherent variability in profile intensity was a significant challenge to the reproducibility
of the method, with subjective calling of signals on the borderline of automated detection.
The variation in fragment intensity is one of the main problems with robust isolate
characterisation by AFLP analysis.
An extensive range of commercially available PCR primer and restriction enzyme
combinations were evaluated for sensitivity against a set of closely related isolates, and
novel combinations targeting hyper-variable regions of the Salmonella genome were also
developed and tested. Genomic digestion with EcoRI and Taq I, followed by amplification
using EcoRI-T and TaqI-0 selective primers was identified as the most discriminatory
approach, and this was used to profile the extensive catalogue of isolates. The AFLP
profiles produced were then compared with known phenotypic characteristics of the
isolates to identify markers correlating with particular traits.
To facilitate identification of unique markers for particular characteristics, a searchable
database was developed at LGC using Microsoft Access. The database allowed both
storage of isolate profiles and comparison of molecular and phenotypic characteristics of
strains, enabling AFLP profiles of unknown samples to be matched with related profiles
already entered into the database. The effectiveness of the database system was
demonstrated in a blind trial, although in contrast to previous studies characterising
smaller numbers of isolates, robust correlation between molecular markers and phage
type or other phenotypic characteristics could not be established.
In conclusion, the AFLP typing approach offers several advantages over currently used
typing methods, including accuracy and precision of fragment sizing, and the suitability of
the profile information for database storage. However, the complex, multi-step nature of
the method can lead to high failure rates, and the relatively high cost of commercial
reagents and equipment are further drawbacks to widespread uptake of the AFLP
approach. Overall, the discriminatory power of the technique was not found to be
sufficiently high to warrant extensive further effort in overcoming the very real drawbacks
of the method, particularly with the advent of newer techniques including proteomic and
microarray methods, and the widespread adoption of PFGE analysis for international
surveillance initiatives. .
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