Final Report Summary - PATHFINDER (Rapid and Reliable Detection of Foodborne Pathogens by Employing Multiplexing Biosensor Technology)
Executive Summary:
Background Access to sufficient, safe & wholesome food has been man's main endeavor from the earliest days of human existence and is one of the basic human rights. One of the main challenges in delivering a safe food supply is the early detection of pathogens to prevent illness due to the consumption of contaminated foods. Pathfinder is an important research project focused on development of innovative means of detecting important pathogenic organisms associated with food poisoning. Molecular binders such as monoclonal antibodies and phage-derived binders were developed and screened for specificity to important foodborne pathogens such as Salmonella and Listeria monocytogenes. The technology platforms were evaluated for their capability of detecting many different pathogens simultaneously by using Salmonella spp., Listeria monocytogenes, and Campylobacter as a model.
Overview of Results:
1. Production of molecular binders A catalog of highly specific monoclonal antibodies to Listeria monocytogenes has been produced and screened by high-throughput screening methods based on microarray and Surface Plasmon Resonance (SPR) [1]. A monoclonal antibody specific for epitopes shared between Campylobacter jejuni and C. coli outer membranes was obtained from Dr. Robert Mandrell (USDA, patent 6551599). Peptide binder specific to Campylobacter was produced at Queen’s University. In addition to the proposed production of antibodies, two libraries of bacteriophages were screened as an alternative binder for Salmonella and Listeria monocytogenes. Several phage peptides demonstrate highly specific binding characteristics to the targets and have been applied to magnetic separation as a detection method [2, 3].
2. Evaluation of biosensors Initially, SPR biosensor was evaluated with a model bacterium, Aacidovorax, whose antibody was readily available at the beginning of the project. Higher detection limit was obtained from the SPR than from the traditional ELISA method. Several strategies, such as different surface chemistries of sensor chips (carboxymethylated dextran CM5 and carboxymethylated C1) and different assay formats (direct, sandwich and subtractive assay), were performed to improve sensitivity of the bacteria detection via SPR [4]. However, no significant improvement was obtained with the bacteria detection. To ensure that this is the case for foodborne pathogen detection, SPR biosensor was used to detect Campylobacter. However, it failed to detect the bacteria even at the concentration of 109 CFU/ml whereas ELISA using the same antibody can detect at the limit of 5x107 CFU/ml. Therefore, we concluded that SPR biosensor is not appropriate for detection of a large molecule like bacteria.
A bead-array technology (MAGPIX) was subsequently explored for bacteria detection. To evaluate the platform while not all antibodies for foodborne pathogens were available, this multiplex microsphere array platform was initially developed to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus) [5]. The multiplex capacity and sensitivity of this platform were better than the standard ELISA method; therefore, it was subsequently employed to develop an equivalent assay for the three foodborne pathogens. With currently available antibodies, the multiplex detection of Salmonella, Listeria, and Campylobacter was successfully developed and ready to be validated with actual food samples from a commercial food company (Betagro, Thailand) during the Returning phase. In addition to using antibodies as a bioreceptor on these platforms, Salmonella-specific bacteriophages were employed to develop a SPR-based assay. Though the detection limit was not low enough to meet the standard required for the food testing industry, the optimization methods to use the bacteriophage as a binder can further be applied for other types of detection [6].
Conclusion:
Pathfinder project has considerably made advancement for the food safety measure by constructing a novel multiplex detection method for foodborne pathogens. Through the process of the development, this project generated several important research milestones. First, different types of binders, monoclonal antibodies and phage-derived peptides, were produced, screened and exploited for their molecular binding capacity. These developed bioreceptors from this project can be further applied to other types of biosensor platforms or other applications beyond imagination. Second, several technology platforms were evaluated. Through these evaluations, multiplex detections for both food and plant pathogens were achieved. The optimization and signal enhancement strategies can also serve as a guideline for similar assay development for these types of biosensors.
Socio-economic Impacts:
The research outputs are of direct significance to food producers/processors, food inspectors and regulatory and testing laboratories. Upon a successful validation of the developed multiplex detection based on bead-array technology, the technology can then be transferred to food producers around the world. This will provide a rapid, accurate, economical testing method for the food industry to reduce the testing cost while increase the credibility for food safety. Moreover, the ability to prevent bacterial contamination in food chain means betterment for public health.
References
[1] R. Charlermroj, M. Oplatowska, M. Kumpoosiri, O. Himananto, O. Gajanandana, C.T. Elliott, et al., Comparison of techniques to screen and characterize bacteria-specific hybridomas for high-quality monoclonal antibodies selection, Anal Biochem, 421(2012) 26-36.
[2] J. Morton, N. Karoonuthaisiri, L.D. Stewart, M. Oplatowska, C.T. Elliott, I.R. Grant, Production and evaluation of the utility of novel phage display-derived peptide ligands to Salmonella spp. for magnetic separation, J Appl Microbiol, 115(2013) 271-81.
[3] J. Morton, N. Karoonuthaisiri, R. Charlermroj, L.D. Stewart, C.T. Elliott, I.R. Grant, Phage display-derived binders able to distinguish Listeria monocytogenes from other Listeria species Plos One, Revised(2013).
[4] R. Charlermroj, M. Oplatowska, O. Gajanandana, O. Himananto, I.R. Grant, N. Karoonuthaisiri, et al., Strategies to improve the surface plasmon resonance-based immmunodetection of bacterial cells, Microchim Acta, 180(2013) 643-50.
[5] R. Charlermroj, O. Himananto, C. Seepiban, M. Kumpoosiri, N. Warin, M. Oplatowska, et al., Multiplex detection of plant pathogens using a microsphere immunoassay technology, PLoS One, 8(2013) e62344.
[6] N. Karoonuthaisiri, R. Charlermroj, M.J. Morton, M. Oplatowska, I.R. Grant, C.T. Elliott, Development of a M13 Bacteriophage-based SPR assay using Salmonella as a case study, Sens Actuators B: Chem, Revised.