Final Report- Pathfinder
Nitsara Karoonuthaisiri
1. Final Publishable Summary Report (2 pages)
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.
Work Package 1
Antibody Development
Work Package 2
Multiplex Selection
Work Package 4
Comparison of multiplex performance
Incoming 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 107 CFU/mL, 5.0-10.0 x 108 CFU/mL and 2.0-6.0 x 108
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.
2.
3.
4.
5.
6.
7.
8.
2.
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.
Morton, J., et al., Phage display-derived binders able to distinguish Listeria monocytogenes
from other Listeria species. PLoS One, 2013. 8(9): p. e74312.
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.
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.
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.
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.
Charlermroj, R., et al., Multiplex detection of plant pathogens using a microsphere
immunoassay technology. PLoS One, 2013. 8(4): p. e62344.
Karoonuthaisiri, N., et al., Bead array for Listeria monocytogenes detection using specific
monoclonal antibodies. Food Control, 2015. 47(0): p. 462-471.
Use and dissemination
Outputs
I. Peer-reviewed publications
Incoming Phase
1. Karoonuthaisiri, N. Charlermroj, R., Teerapornpuntakit, J., Kumpoosiri, M.,
Himananto, O., Grant, I.R., Gajanandana, O., Elliott, C.T., Production and
characterization of specific Listeria monocytogenes monoclonal antibodies
and their applications in two immunochemical platforms. (submitted)
2. Karoonuthaisiri, N. Charlermroj, R., Morton, M.J., Oplatowska, M., Grant,
I.R., Elliott, C.T., Development of a M13 Bacteriophage-based SPR detection
assay using Salmonella detection as a case study (revised Sens. Actuators B:
Chem)
3. Morton, M.J., Karoonuthaisiri, N., Charlermroj, R., Stewart, L.D., Elliott,
C.T., Grant, I.R. 2013. Phage display-derived binders able to distinguish
Listeria monocytogenes from other Listeria species. Plos One (accepted)
4. Morton, M.J., Karoonuthaisiri, N., Stewart, L.D., Oplatowska, M., Elliott,
C.T., Grant, I.R. 2013. Production and evaluation of the utility of novel phage
display-derived peptide ligands to Salmonella spp. for magnetic separation. J
Appl Microbiol 115: 271—281. doi: 10.1111/jam.12207
5. Charlermroj, R., Oplatowska, M., Gajanandana, O., Himananto, O., Grant,
I.R., Karoonuthaisiri, N., Elliott, C.T. 2013. Strategies to Improve the
Surface Plasmon Resonance-Based Immmunodetection of Bacterial Cells.
Microchimica Acta 180(7): 643-650. doi: 10.1007/s00604-013-0975-x
6. Charlermroj, R., Himananto, O., Seepiban, C., Kumpoosiri, M., Warin, N.,
Oplatowska, M., Gajanandana, O., Grant, I.R., Karoonuthaisiri, N., Elliott,
C.T. 2013. Multiplex Detection of Plant Pathogens Using a Microsphere
Immunoassay
Technology.
PloS
ONE
8(4):e62344.
doi:10.1371/journal.pone.0062344
7. Charlermroj, R., Oplatowska, M., Kumpoosiri, M., Himananto, O.,
Gajanandana, O., Elliott, C., Karoonuthaisiri, N.* 2012. Comparison of
techniques to screen and characterize bacteria-specific hybridomas for high
quality monoclonal antibodies selection. Analytical Biochemistry 421(1):2636. doi: 10.1016/j.ab.2011.10.005
Returning Phase
8. Wang, J., Morton, M., Elliott, C., Karoonuthaisiri, N., Segatori, L., Biswal,
S. L. 2014. Rapid detection of pathogenic bacteria and screening of phagederived peptides using microcantilevers. Anal Chem. 86 (3): 1671–1678
9. Karoonuthaisiri, N.*, Charlermroj, R., Teerapornpuntakit, J., Kumpoosiri,
M., Himananto, O., Grant, I.R., Gajanandana, O., Elliott, C.T. Bead array for
Listeria monocytogenes detection using specific monoclonal antibodies. Food
Control 47 (2015) 462-471
II. Workshop, seminar and conferences
Phase
Incoming
Date
29-30
September
2011
12 March
2012
13 March
2012
24 February
2012
1-2 May
2012
12-13 June
2012
19 June
2012
18-19 June
2013
Name
Microarray World
Congress
Location
San Francisco,
USA
Responsibility
Speaker
FP7/FP8 People
Programme Workshop
FP7 Cooperation-Food
and Environment Info
Day
How to write an FP7
Marie Curie Proposal
Safefood Microbial
Networks Conference
Immunomagnetic bead
array training (Luminex
Training)
BIOTEC,
Thailand
BIOTEC,
Thailand
Organizer,
Speaker
Organizer,
Speaker
Queen’s
University, UK
Belfast, UK
Speaker
Institute for
Global Food
Security,
Queen’s
University, UK
Bonn, Germany
Organizer
Institute for
Global Food
Security,
Queen’s
University, UK
Organizer
Challenges and
opportunities of
International Cooperation
in Horizon 2020
Microarray fabrication
using Piezoeletric
microarray spotter
(Scienion Training)
Speaker
Speaker
1-2 Aug
2013
Cartridge assembly for a
multiplexed fluorescence
immunoassay system
(MBio Array Training)
Institute for
Global Food
Security,
Queen’s
University, UK
Returning 22 Oct 2013 Risk assessment and food National Science
safety in chicken
and Technology
industry: farm to fork.
Development
Agency
11-13 Dec
ASEAN+ 2013 The 2nd
Chiang Rai,
2013
Regional Symposium on Thailand
Biosensors,
Biodiagnostics and
Biochips
2-3 Apr
The 4th International
Bangkok,
2014
Biochemistry and
Thailand
Molecular Biology
Conference.
21-23 May International Conference Bangkok,
2014
on Discrete Mathematics Thailand
and Applied Sciences
29-30 Sep
International BioScience Phuket Thailand
2014
Conference
III. PhD Students
Mr. Ratthaphol Charlermroj, graduated with PhD on 1 July 2013
Mrs. Mary Josephine Morton, graduated with PhD in Winter 2013
Organizer and
trainer
Speaker
Keynote speaker
Invited Speaker
Invited Speaker
Invited Speaker