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Phase I research will complete five primary technical objectives with the end goal being demonstration of gene-based, real-time detection of pathogens in agricultural settings utilizing the detection kits and a handheld, portable detection platform developed by Diagenetix, Inc. The pathogens targeted in this project include Salmonella enterica on almonds, Candidatus Liberibacter in tissues of citrus plants and citrus psyllids, and Erysiphe necator spores in the vineyard, and Botrytis cinerea in grape tissue. However, the kits and platform are not limited to these and can be adapted to all pathogens. Two significant technical objectives involve the development of ready-to-use reagent kits using Diagenetix Inc.'s patent pending Assimilating Probes. The use of the Assimilating Probe technology ensures sequence specific amplification and an internal control, confirming the test and kit's integrity. Performance requirements and specifications will first be determined and then formulations will be tested to ensure requirements are met. The primary performance requirements involve the accurate and sensitive detection of the target pathogens. The development of these ready-to-use kits and completion of the two objectives will be completed by the end of Month 2 of the project period. In addition to the reagent kits, complimentary handheld, portable instruments will also be manufactured. Performance requirements will be defined to also ensure accuracy and sensitivity of the detection kits. The handhelds will also seek to meet size, weight, durability, and power use specifications. The instruments will be completed at the end of Month 2 of the project period. In Month 3, training will be provided to representatives of the phytopathology community. The training will include sample preparation and use of the kits and instrument. The remaining performance period will be used to field-test the kits, instrument, and associated sample preparation methodology. This testing will take place by the representatives and will serve to provide feedback for improvements and establish a baseline data set to compare the developed detection platform relative to existing lab-based solutions.
<p>NON-TECHNICAL SUMMARY: <br/>Intensification of resource use and other global changes have resulted in emergence of a variety of new diseases and increasing virulence of existing diseases, like Salmonella. These pests and disease organisms are ever more efficiently spread by traffic in goods and people throughout the world, and pose major risks to agriculturally sensitive areas and human health. Exclusion of exotic diseases from US agriculture is of primary importance to the biosecurity of the nation, and management of established diseases is becoming ever more important to agricultural industries with low margins which are increasingly challenged to meet global needs for food with limited resources. The primary objective of this project is to demonstrate simple new technologies to enable rapid field detection of pathogens with user friendly data management and reporting to
facilitate management of a wide variety of disease organisms of importance to agriculture. Partners in the research community have been selected to demonstrate these technologies for a variety of important pathogens including the citrus greening organism (the most destructive pathogen of citrus), Salmonella (a bacteria which causes the highest incidence of food-borne illness in the US and globally), and fungal pathogens which have severe economic impacts on fruit. The new technologies, which are based on isothermal gene-based detection, enable rapid, sensitive, and selective detection of important pathogens with simple, inexpensive hardware operated by personnel with limited diagnostic experience. These characteristics are increasingly important for safeguarding the biosecurity and competitiveness of agriculture in the US. These technologies are poised to capture significant market share
in the food and agricultural diagnostics industry.
<p>APPROACH: <br/>Primers and Assimilating Probes will be tested and optimized using representative samples spiked with extracts of target pathogen DNA at varying concentrations to determine sensitivity, accuracy, and speed. Use of representative sample material will help determine if there are inhibitors to the reaction in the sample material. The sample material includes psyllid and citrus plant material for Candidatus Liberibacter, rinsate of raw almonds for Salmonella enterica, mixed particulates captured from air for E. necator, and grape juice for B. cinerea. The design, testing, and optimization of primers and probes will be conducted in a laboratory setting, using a real-time PCR machine. It will then be also confirmed in the lab using the developed handheld platform. It is anticipated there will be no difference in results between the results from a real-time PCR
machine and the handheld platform. The primers and Assimilating Probes will then be combined with other reagents to formulate a ready-to-use mix. The mix will then be once again tested on a real-time PCR machine and the handheld platform to ensure consistency and accuracy. Upon completion of the lab-based assessment, the kits and portable instrument will be given to agents of the USDA for in-field testing. The agents will run in parallel testing for the target pathogens using the provided method, kit, and instrument and using an existing methodology, in most cases real-time PCR will be used.
<p>PROGRESS: 2012/05 TO 2013/05 <p>Target Audience: <br/>The target audience for this project was researchers, regulatory bodies, and agricultural producers and processors. During the reporting period Diagenetix reached researchers by collaborating with USDA-ARS researchers. in July 2012, Diagenetix hosted a workshop in Honolulu for the phase I collaborators from USDA-ARS, including Dr. Manjunath Keremane (Riverside, CA), Mr. Eric Jackson (Albany, CA), and Ms. Tara Neill and Ms. Lindsey Thiessen of Dr. Mahaffee’s group (Corvallis, OR). At the workshop, the participants were given hands-on training using our diagnostic platform. Participants were also invited to share their experiences related to the specific pathosystems they work on, and their approaches to sample capture and preparation. Several simple and effective technologies were demonstrated, including the
rotary spore traps developed by the group from Corvallis. The workshop also provided an opportunity for all of the participants to strategize about the project, and the key objectives were prioritized based on the most recent analysis of the needs and constraints faced by the respective collaborators. We then manufactured 12 platforms and provided them to our USDA-ARS collaborators for in-field testing and demonstration. Our collaborators demonstrated the platform and assays are suitable for rapid detection of target organisms in the field. They also attended industry conferences where they were able to share our platform and work with agricultural producers and processors. We additionally demonstrated our platform for the FDA. They then invited us to participate in a field trial with their mobile lab. Ourselves and one of our USDA-ARS collaborators participated in the trial in Salinas,
CA in September 2012. There we demonstrated our platform was appropriate for in-field use. In summary, we directly and indirectly reached our target audience during the reporting period. <p>Changes/Problems: <br/>Two major challenges were encountered. The first involves the creation of ready-to-use kits. This was more challenging and time consuming than originally believed. As such, we sought help from outside experts and requested a no-cost extension to ensure that other goals and milestones were not impacted. We currently have a method to compose ready-to-use kits. However, this method relies upon a third party vendors to lyphlilize each chemical component separately. We have purposed work in our Phase II to be able to lypholize reagents in one tube, making post-process handling of the kit easier as total material handling is significantly larger relative to separate lyphilization. The second
major challenge encountered was the regarding the manufacturing of the handheld device. This challenge was due to machinery malfunctions at the sub-contractor's facilities. This resulted in delays in prototyping and manufacturing of components. To mitigate risks of the delays, we sourced some additional parts from a third party. These served as a back-up. Ultimately, we were able to manufacture some components using our sub-contractor's facilities and also utilized some components from the third party. What opportunities for training and professional development has the project provided? One primary goal was to provide training to USDA-ARS collaborators. A workshop was conducted to facilitate this training. How have the results been disseminated to communities of interest? The results of the work to date has been shared via our collaborators. They have shared the work with
members in their representative industry as well as those at USDA-APHIS. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported