"For simultaneous growth and detection of Salmonella spp., E. coli O157:H7, and L. monocytogenes, we formulated a selective enrichment broth SEL (Salmonella, E. coli O157:H7, and Listeria)..."

Multipathogen screening using immunomicroarry

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

Project Report 2007 - 2008

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Project Rationale

Antibody-based methods for detecting pathogenic foodborne bacteria are used widely and are regarded as rapid and efficient. Application of conventional antibody-based assays and further adaptation in modern biosensor tools show promise for rapid detection. Most assays are developed for detection of a single target pathogen or toxin. As a result, these methods can be costly when testing for multiple pathogens from a single product because separate assay methods are required. In addition, a large laboratory space is required to perform separate tests for each target pathogen because separate enrichment media and procedures are needed for each pathogen detection method. The development of a single test capable of detecting multiple pathogens enriched in a single enrichment media would reduce costs and yield quick results. This technology would also benefit regulatory agencies in the evaluation of food products for key food pathogens.

In the past two decades, several rapid detection methods, such as antibody-based, nucleic acid-based, and biochemical-based methods, were developed. Even though these methods have shortened analysis times, it still takes time for selective enrichment of samples before conventional rapid detection methods can be employed. Antibody-based methods, such as enzyme-immunoassay, require a minimum of 106 CFU/ml for cell detection. To achieve that cell level, it is important to use proper enrichment media for detection of foodborne pathogens.

Cell injury or stress encountered during food processing may affect cell numbers. The presence of selective agents in media can delay growth and recovery of stressed or injured cells. Thus, currently used selective enrichment media may not be suitable for use with modern detection methods. Furthermore, methodologies that detect multiple pathogens with a single device are preferred. Therefore, a medium that can support selective enrichment of multiple pathogens is desirable.

Project Objectives

  • Develop a microarray assay in 96-well plate and glass slide using sandwich immunoassay for three pathogens: Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes
  • Optimize growth and enrichment of these pathogens (healthy or stressed) spiked in model food samples in a selective enrichment broth for use with microarray.
  • Evaluate performance of the Pathogen Enrichment Device (PED).

Project Highlights

For simultaneous growth and detection of Salmonella spp., E. coli O157:H7, and L. monocytogenes, we formulated a selective enrichment broth SEL (Salmonella, E. coli O157:H7, and Listeria) and found it to be suitable for enrichment of all these target pathogens in spiked ready-to-eat deli turkey and salami. In addition, when we evaluated the performance of SEL for its ability to detect pathogens by immunoassay, we found that target pathogens grown in SEL gave a stronger response than when they were grown on other media. The global protein expression profiles of the three pathogens in SEL were significantly greater than their respective selective enrichment broths. Proteomic analysis further revealed the presence of a unique protein in bacteria when grown on SEL.