Rapid Phage-based Method for the Detection of Pathogens in Food

The goal of the project is to develop and evaluate a bacteriophage-based system for the simultaneous capture and detection of food-borne pathogens in food and environmental samples. The system will use immobilized host-specific reporter bacteriophage to concentrate the target bacteria on the surface of magnetic beads and, following bacterial infection by the phage, a bioluminescent or fluorescent signal will appear with intensity proportional to the initial number of bacteria in the sample.
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For constructing reporter bacteriophages, two different reporter systems will be used. In the first, lux genes will be introduced into the phage genome using protocols described previously. After the infection the developing bioluminescence will be monitored and correlation between initial cell number and in vivo bioluminescence will be established. The second reporter phage will be constructed by fusion of GFP gene with phage head protein gene, thus providing release of phage particles labeled with GFP after infection. Immobilization of phages will be performed by introduction of biotin or biotin-binding peptides onto the bacteriophage head. This will be done both by chemical modification of amino-or carboxyl-groups of head or by fusion of head protein with the biotin-binding peptide according to a published experimental protocol. Biotinylated bacteriophages will be immobilized on streptavidin-coated solid surface resulting in formation of a biosorbent.