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Rapid and Reliable Detection of Foodborne Pathogens by Employing Multiplexing Biosensor Technology

Final Report Summary - PATHFINDER (Rapid and Reliable Detection of Foodborne Pathogens by Employing Multiplexing Biosensor Technology)

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. The project was proposed into two phases: Incoming phase and Returning phase as depicted in Fig. 1. This report focuses only on Work Package 4, which was conducted during the Returning phase.

Fig. 1. Overview of resarch program for Pathfinder

Overview of Results
Previously, during the incoming phase, 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]. In addition to the proposed production of antibodies, two libraries of bacteriophages were screened for an alternative binder for Salmonella and Listeria monocytogenes. Several phage peptides demonstrated highly specific binding characteristics to the targets and have been applied to magnetic separation as a detection method [2, 3]. These specific binders and other available binders were employed to evaluate different biosensor platforms such as SPR [4, 5], microcantilever [6], and a bead-array technology (MAGPIX) [7]. After such evaluation, a bead-array technology was the most appropriate method due to its multiplex capacity and sensitivity which were better than the standard ELISA method.
During the returning phase, the bead-array technology was used to perform multiplex detection of Salmonella spp., Listeria monocytogenes, and Campylobacter spp. in actual food samples (roasted chicken barbecue with honey, grilled black pepper chicken, and raw chicken marinated in sesame sauce) [8].
To simulate foodborne pathogens contamination, each pathogen was inoculated into the food samples at 0, 1, and 10 colony forming unit (CFU). The samples were subsequently cultured according to the international organization for standardization (ISO 10272, 11290, and 6579 for Campylobacter spp., Listeria monocytogenes, and Salmonella spp., respectively). Samples from each culture protocol were mixed and tested at 24, 48 and 72 hours by the bead-array method and a conventional plate-count protocol to enumerate the bacteria number. The results showed that the bead-array method was able to detect even at 1 CFU after being cultured for 24 hours in case of Salmonella spp. and 48 hours in case of Listeria monocytogenes and Campylobacter spp. From the plate-count method of the 24-hr culture of Salmonella spp and the 48-hr cultures of Listeria monocytogenes and Campylobacter spp., 6.0- 40.0 x 10^7 CFU/mL, 5.0-10.0 x 10^8 CFU/mL and 2.0-6.0 x 10^8 CFU/mL were found, respectively.

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. In addition, the developed bead array method has been demonstrated to be able to detect Campylobacter spp., Listeria monocytogenes, and Salmonella spp. contaminated in various actual food samples using the conventional enrichement methods according to the international organization for standardization.

Socio-economic Impacts
The research outputs are of direct significance to food producers/processors, food inspectors and regulatory and testing laboratories. A successful validation of the developed multiplex detection based on bead-array technology is to be transferred to food producers. 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. Charlermroj, R., et al., Comparison of techniques to screen and characterize bacteria-specific hybridomas for high-quality monoclonal antibodies selection. Anal Biochem, 2012. 421(1): p. 26-36.
2. Morton, J., et al., Phage display-derived binders able to distinguish Listeria monocytogenes from other Listeria species. PLoS One, 2013. 8(9): p. e74312.
3. Morton, J., et al., Production and evaluation of the utility of novel phage display-derived peptide ligands to Salmonella spp. for magnetic separation. J Appl Microbiol, 2013. 115(1): p. 271-81.
4. Charlermroj, R., et al., Strategies to improve the surface plasmon resonance-based immmunodetection of bacterial cells. Microchimica Acta, 2013. 180(7-8): p. 643-650.
5. Karoonuthaisiri, N., et al., Development of a M13 bacteriophage-based SPR detection using Salmonella as a case study. Sensors and Actuators B-Chemical, 2014. 190: p. 214-220.
6. Wang, J.H. et al., Rapid Detection of Pathogenic Bacteria and Screening of Phage-Derived Peptides Using Microcantilevers. Analytical Chemistry, 2014. 86(3): p. 1671-1678.
7. Charlermroj, R., et al., Multiplex detection of plant pathogens using a microsphere immunoassay technology. PLoS One, 2013. 8(4): p. e62344.
8. Karoonuthaisiri, N., et al., Bead array for Listeria monocytogenes detection using specific monoclonal antibodies. Food Control, 2015. 47(0): p. 462-471.