"If a single test is available that can detect multiple pathogens enriched in a single enrichment broth, this will not only reduce cost but also will be convenient and provide results in a short period of time."

Multipathogen screening using immunomicroarry

Investigator: Arun K. Bhunia (Department of Food Science)

Project Report 2005 - 2006

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Project Rationale

Antibody-based methods are widely used for the detection of most pathogenic bacteria, and are regarded as rapid and efficient. Their application to a conventional ELISA assay and further adaptation in modern biosensor tools shows promise in rapid detection. Most assays are developed for detection of a single target pathogen or toxin. Therefore, it is very expensive to test for multiple pathogens from a single product because separate assay methods need to be used, thus adding cost and labor per test. Also, large laboratory space is required to perform separate tests for each target pathogen because separate enrichment reagents and procedures need to be applied for different pathogens. If a single test is available that can detect multiple pathogens enriched in a single enrichment broth, this will not only reduce cost but also will be convenient and provide results in a short period of time. This would also benefit the regulatory agencies when evaluating food products for key food pathogens.

In the last decade, several rapid detection methods, such as antibody-based, nucleic acid-based, and biochemical-based, were developed. Even though these methods have shortened the analysis time compared to the conventional detection method, still we have to allow time for selective enrichment of samples prior to employing rapid detection methods. An antibody-based method such as ELISA requires at least 106 CFU/ml for detection. Thus, to achieve that level of cells, it is important to use proper enrichment media for detection of foodborne pathogens. Furthermore, cell injury or stress encountered during food processing may affect cell numbers.

Additionally, selective agents in media can delay growth and recovery of stressed or injured cells. Thus, currently used selective media may not be suitable for use with modern detection methods. Furthermore, current trends emphasize the detection of multiple targets with a single device. To achieve that, a medium that can allow selective enrichment of multiple pathogens is desirable.

Project Objectives

The overall objective of this project is to develop immunomicroarray for concurrent detection of viable cells of three pathogens; L. monocytogenes, E. coli O157:H7 and Salmonella enterica.

The specific objectives are:

  • Development of microarray assay in 96-well plate and glass slide using sandwich immunoassay for three pathogens.
  • Optimizing growth and enrichment of three pathogens (healthy or stressed) spiked in model food samples in a selective enrichment broth for use with microarray.

Project Highlights

Our group has developed a selective enrichment medium for simultaneous growth and detection of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella. The media, designated SEL (Salmonella, E. coli, Listeria) was formulated using Buffered Listeria Enrichment Broth (BLEB) base and various combinations of antibiotics: acriflavine, cycloheximide, fosfomycin and nalidixic acid. Initial testing indicated that this medium supports the growth of E. coli O157:H7, L. monocytogenes and S. enteritidis well, and the growth rate of each is comparable to the respective selective enrichment broth such as modified EC medium for E. coli, RV for Salmonella and FB for Listeria.