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Improvement and Validation of a Novel Protein Microarray Assay for Salmonella Serotyping


A rapid, cost-effective and easy-to-use protein (antibody) microarray assay has been successfully developed for Salmonella serotyping by the Kauffmann-White scheme in our previous project. The assay involves immobilization of Salmonella antibodies onto epoxy activated glass slides, labelling of Salmonella cells with the fluorescent dye Eosin Y, capturing Salmonella cells by the antibodies, and detection of the fluorescent signal using a microarray scanner. A prototype antibody array was constructed for identification of 20 commonly identified and clinically important Salmonella serovars. The antibody array was able to detect multiple Salmonella O antigens, and both phase 1 and 2 flagella antigens simultaneously, and thus allowed correct one-step identification of the serovar. The assay was evaluated for Salmonella serotyping using 117 target and 73 non-target Salmonella strains belonging to 58 serovars. The microarray profiles allowed correct serovar identification of 86 target strains, and correct identification of O and most of the phase antigens for an additional 30 target strains. The assay also allowed exclusion of the 73 non-target strains from the 20 target serovars. Because of its speed, accuracy, low cost, and ability to identify both flagellar antigens simultaneously, the antibody microarray-based assay is a promising alternative to the current slide agglutination method for Salmonella serotyping. However, the assay needs further optimization, validation under applied conditions and standardized data analysis method/tool for its implementation in diagnostic laboratories.
In this project, we will improve and validate the system to make it operational by diagnostic labs. More specific antibodies and additional antibodies will be evaluated and included on the microarray to minimize impact of cross-reactions, and to allow exclusion of closely-related serovars. A customized software program will be created to facilitate data analysis, interpretation and reporting. Sufficient antibody arrays will be produced and the system will be validated using at least 1000 Salmonella strains. The validation will be conducted side-by-side with the standard slide agglutination method under applied conditions in collaboration with the OIE Reference Laboratory for Salmonellosis, Public Health Agency of Canada (PHAC).

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Expected Impact of Project Outcomes on Food Safety in Ontario:
Salmonella consists of over 2500 serovars. Serotyping is the most important universal typing method for characterization of Salmonella isolates, and involves more than 250 antisera for identification of all serovars. The current Salmonella serotyping method only allows for detection of a single antibody-antigen reaction at a time. A minimum of three to six antibody-antigen reactions are needed for recognition of a particular Salmonella serovar. The number of reactions required can be many times greater if a less common serovar is tested. The assay consumes high volumes of reagents, and takes three days to two weeks to provide an answer.
Successful completion of our proposed project will result in an improved and validated protein microarray-based test that can be used for rapid and cost-effective serotyping of Salmonella in a single test. This will allow testing laboratories and government inspection staff to conduct risk assessment and outbreak studies in a cost-effective and timely manner in support of monitoring and surveillance activities and enforcement of regulatory programs. The method developed in this project can be extended to serotyping of other food-borne pathogens such as Campylobacter.
<P> For more information, please visit the <a href="; target="_blank">Ontario Ministry of Agriculture, Food & Rural Affairs (OMAFRA) Food Safety Research Program</a>.

Chen, Shu
University of Guelph
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