Performing Department
(N/A)
Non Technical Summary
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.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
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.
Project Methods
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.