Diagnostic Tests and their Application in the Management of Soil- and Water-borne Oomycete Pathogen Species

Oomycete diseases cause significant losses across a broad range of crop and aquaculture commodities worldwide. These losses can be greatly reduced by disease management practices steered by accurate and early diagnoses of pathogen presence. Determinations of disease potential can help guide optimal crop rotation regimes, varietal selections, targeted control measures, harvest timings and crop post-harvest handling. Pathogen detection prior to infection can also reduce the incidence of disease epidemics. Classical methods for the isolation of oomycete pathogens are normally deployed only after disease symptom appearance. These processes are often-time consuming, relying on culturing the putative pathogen(s) and the availability of expert taxonomic skills for accurate identification; a situation that frequently results in either delayed application, or routine ‘blanket’ over-application of control measures. Increasing concerns about pesticides in the environment and the food chain, removal or restriction of their usage combined with rising costs have focussed interest in the development and improvement of disease management systems. To be effective, these require timely, accurate and preferably quantitatve diagnoses. A wide range of rapid diagnostic tools, from point of care immunodiagnostic kits to next generation nucleotide sequencing have potential application in oomycete disease management. Here we review currently-available as well as promising new technologies in the context of commercial agricultural production systems, considering the impacts of specific biotic and abiotic and other important factors such as speed and ease of access to information and cost effectivenes

Development of multiplex detection for plant pathogens using antibody array technology in a multiwell-plate format, surface plasmon resonance (SPR) and bead array technology

The three different platform candidates were antibody microarray, surface Plasmon resonance (SPR) and bead array. Another objective of this project was to explore other applications of these platforms, and the selected example application was to explore other applications of these platforms for hybridoma screening. Since antibodies against plant pathogens have previously been produced, characterized and used in development of the three different platforms, to examine the possibility of applying the platforms to facilitate the screening of hybrid om a, pilhogenic foodbome bacterium Listeria monocytogenes was used as a model study. In summary, antibody array and bead array technologies were demonstrated to be able to detect multiple plant pathogens at the same time. Both were shown to be able to detect the pathogens in the real samples from fields. making them good candidates for plant industries. On the other hand, despite considerable effort to incr¢ase the sensiritivity of SPR, lower limit of detection could not be achieved. This courd be an intrinsic limitation of this method for detecting a whole cell where the size of the analytic is bigger than usual SPR analytes such as small compounds. From these technologies. an antibody array was selected for hybridoma screening and was shown to be effective, albeit with lower sensitivity compared to ELISA. The antibody array proved to be a faster and simpler method to screen hundreds of hybricioma 3 clones. A comnbination of biosensor tcchnology ensures good quality screening for hybridoma clones as demonstrated in this study.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Phage Display-Derived Binders Able to Distinguish <em>Listeria monocytogenes</em> from Other <em>Listeria</em> Species

The objective of this study was to produce phage display-derived binders with the ability to distinguish Listeria monocytogenes from other Listeria spp., which may have potential utility to enhance detection of Listeria monocytogenes. To obtain binders with the desired binding specificity a series of surface and solution phage-display biopannings were performed. Initially, three rounds of surface biopanning against gamma-irradiated L. monocytogenes serovar 4b cells were performed followed by an additional surface biopanning round against L. monocytogenes 4b which included prior subtraction biopanning against gamma-irradiated L. innocua cells. In an attempt to further enhance binder specificity for L. monocytogenes 4b two rounds of solution biopanning were performed, both rounds included initial subtraction solution biopanning against L. innocua. Subsequent evaluations were performed on the phage clones by phage binding ELISA. All phage clones tested from the second round of solution biopanning had higher specificity for L. monocytogenes 4b than for L. innocua and three other foodborne pathogens (Salmonella spp., Escherichia coli and Campylobacter jejuni). Further evaluation with five other Listeria spp. revealed that one phage clone in particular, expressing peptide GRIADLPPLKPN, was highly specific for L. monocytogenes with at least 43-fold more binding capability to L. monocytogenes 4b than to any other Listeria sp. This proof-of-principle study demonstrates how a combination of surface, solution and subtractive biopanning was used to maximise binder specificity. L. monocytogenes-specific binders were obtained which could have potential application in novel detection tests for L. monocytogenes, benefiting both the food and medical industries

A rapid colorimetric lateral flow test strip for detection of live

Specific antibodies are essential components of immunoassay, which can be applied for the detection of pathogens. However, producing an antibody specific to live bacterial pathogens by the classical method of immunizing animals with live pathogens can be impractical. Phage display technology is an effective alternative method to obtain antibodies with the desired specificity against selected antigenic molecules. In this study, we demonstrated the power of a microarray-based technique for obtaining specific phage-derived antibody fragments against Salmonella, an important foodborne pathogen. The selected phage-displayed antibody fragments were subsequently employed to develop a lateral flow test strip assay for the detection of live Salmonella. The test strips showed specificity to Salmonella Enteritidis without cross-reactivity to eight serovars of Salmonella or other bacteria strains. The test strip assay requires 15 min, whereas the conventional biochemical and serological confirmation test requires at least 24 h. The microarray screening technique for specific phage-based binders and the test strip method can be further applied to other foodborne pathogens

Implementation of microfluidic sandwich ELISA for superior detection of plant pathogens.

Rapid and economical screening of plant pathogens is a high-priority need in the seed industry. Crop quality control and disease surveillance demand early and accurate detection in addition to robustness, scalability, and cost efficiency typically required for selective breeding and certification programs. Compared to conventional bench-top detection techniques routinely employed, a microfluidic-based approach offers unique benefits to address these needs simultaneously. To our knowledge, this work reports the first attempt to perform microfluidic sandwich ELISA for Acidovorax citrulli (Ac), watermelon silver mottle virus (WSMoV), and melon yellow spot virus (MYSV) screening. The immunoassay occurs on the surface of a reaction chamber represented by a microfluidic channel. The capillary force within the microchannel draws a reagent into the reaction chamber as well as facilitates assay incubation. Because the underlying pad automatically absorbs excess fluid, the only operation required is sequential loading of buffers/reagents. Buffer selection, antibody concentrations, and sample loading scheme were optimized for each pathogen. Assay optimization reveals that the 20-folds lower sample volume demanded by the microchannel structure outweighs the 2- to 4-folds higher antibody concentrations required, resulting in overall 5-10 folds of reagent savings. In addition to cutting the assay time by more than 50%, the new platform offers 65% cost savings from less reagent consumption and labor cost. Our study also shows 12.5-, 2-, and 4-fold improvement in assay sensitivity for Ac, WSMoV, and MYSV, respectively. Practical feasibility is demonstrated using 19 real plant samples. Given a standard 96-well plate format, the developed assay is compatible with commercial fluorescent plate readers and readily amendable to robotic liquid handling systems for completely hand-free assay automation

Data_Sheet_2_A rapid colorimetric lateral flow test strip for detection of live Salmonella Enteritidis using whole phage as a specific binder.DOCX

Specific antibodies are essential components of immunoassay, which can be applied for the detection of pathogens. However, producing an antibody specific to live bacterial pathogens by the classical method of immunizing animals with live pathogens can be impractical. Phage display technology is an effective alternative method to obtain antibodies with the desired specificity against selected antigenic molecules. In this study, we demonstrated the power of a microarray-based technique for obtaining specific phage-derived antibody fragments against Salmonella, an important foodborne pathogen. The selected phage-displayed antibody fragments were subsequently employed to develop a lateral flow test strip assay for the detection of live Salmonella. The test strips showed specificity to Salmonella Enteritidis without cross-reactivity to eight serovars of Salmonella or other bacteria strains. The test strip assay requires 15 min, whereas the conventional biochemical and serological confirmation test requires at least 24 h. The microarray screening technique for specific phage-based binders and the test strip method can be further applied to other foodborne pathogens.</p

Multiplex Detection of Plant Pathogens Using a Microsphere Immunoassay Technology

Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays 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). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection

Data_Sheet_1_A rapid colorimetric lateral flow test strip for detection of live Salmonella Enteritidis using whole phage as a specific binder.DOCX

Specific antibodies are essential components of immunoassay, which can be applied for the detection of pathogens. However, producing an antibody specific to live bacterial pathogens by the classical method of immunizing animals with live pathogens can be impractical. Phage display technology is an effective alternative method to obtain antibodies with the desired specificity against selected antigenic molecules. In this study, we demonstrated the power of a microarray-based technique for obtaining specific phage-derived antibody fragments against Salmonella, an important foodborne pathogen. The selected phage-displayed antibody fragments were subsequently employed to develop a lateral flow test strip assay for the detection of live Salmonella. The test strips showed specificity to Salmonella Enteritidis without cross-reactivity to eight serovars of Salmonella or other bacteria strains. The test strip assay requires 15 min, whereas the conventional biochemical and serological confirmation test requires at least 24 h. The microarray screening technique for specific phage-based binders and the test strip method can be further applied to other foodborne pathogens.</p