Microarray for Salmonella serotyping

Salmonella is one of the major bacterial pathogens that cause food-borne infections worldwide (Herikstad et al., 2002). Numerous typing methods have been developed to trace salmonellosis outbreaks to the contaminated source and to delineate the epidemiology of Salmonella infections. The most commonly used approach to characterise Salmonella strains is serotyping of the phase 1 and 2 flagellar proteins (the H1 and H2 antigens, respectively) and the O-specific polysaccharide (O antigen) in the microbe`s lipopolysaccharide-containing outer membrane. However, serotyping is very time-consuming, requires the use of more than 250 specific sera and consequently this methodology is not available in many laboratories. Furthermore, commercial antisera are not always available and their quality may often vary. The development of molecular methods providing similar or even higher power of discrimination and allowing for rapid high-throughput identification of Salmonella subspecies and serotypes is urgently needed. Molecular methods are also easier to standardise due to the higher stability and easier production of nucleic acid probes compared to antisera. Microbial diagnostic microarrays (also termed identification arrays, genotyping arrays, phylogenetic oligonucleotide arrays or phylochips) enable the parallel detection and identification of a large number of microbes. Microbial diagnostic microarrays is a dynamically developing novel technical field of microbiology. The last two years witnessed a first wave of successful adaptations of the microarray technology to microbial diagnostics. While developed more or less independently in different research labs, these first generation methodologies share a number of common shortcomings. Most notably, they are limited in sensitivity, specificity, speed and resolution power. Some of the strong potential of the technology has already been demonstrated with these first generation microarrays. However, to realise the full potential, especially in the fields of clinical and food microbiology, several improvements are to be made. Locked nucleic acid (LNA) is a novel DNA/RNA homologue with enhanced hybridisation potential. LNA chemistry is fully compatible with DNA/RNA chemistry, thus allowing for the improvement of existing DNA or RNA oligoprobes by replacing individual residues to LNA. Evanescent field scanning (EF) technology enables the on-line monitoring of hybridisation events on the surface of a microarray by focusing the layer of detection to a narrow layer, excluding the signal from unbound target molecules. The parallel, on-line monitoring of individual hybridisation events enables the acquisition of hybridisation signal for each individual probe. Here we propose a project for the development of an improved methodology of microbial diagnostic microarrays, based on our existing knowledge (gained during the development and application of a first generation methodology) and LNA technology, supported in the development phase by evanescent field scanning. This will be applied for the development of a microarray that allows high-throughput, easily standardisable identification of Salmonella serotypes. The developed microarray will be validated using Salmonella isolates from reference laboratories across Europe.