Progress 09/01/18 to 04/30/20

Outputs
Target Audience:In Phase I of this project, Agdia collaborated with virologists in the plant science community to validate an assay for Tobacco rattle virus detection. Through these collaborations, we were able to demonstrate the efficacy of a TRV isothermal assay for screening potato and ornamental crops. We are currently expanding our reach to end-users (diagnosticians, breeders, producers) with the commercial launch of the AmplifyRP® XRT for TRV product on June 15, 2020. The commercialization plan for the miniature fluorometer, developed through consultations with Dr. Calcaterra and Larta Institute, provides a roadmap for future outreach. We plan on working closely with our customers on evaluation of the prototype device as development continues in Phase II and beyond. Changes/Problems:Design and development of a molecular assay for the detection of Tobacco rattle virus and an endogenous control was remarkably successful; at the conclusion of Phase I, all of the technical objectives were achieved and validation was completed. The second component of this two-part effort towards a low-cost detection platform was the development of a miniaturized fluorometric device. Early data produced from the original optical design indicated that sensitivity improvements were needed to better align with higher-cost equipment that is currently available. In response, Agdia redesigned the optical system for a handheld prototype instrument that can detect the virus at concentrations found in infected plants. Further research is needed to configure the optimal arrangement of component parts to allow for greater sensitivity and precision in measurement. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Results of the TRV AmplifyRP XRT assay development were presented at both domestic and international plant science conferences. The 2019 Plant Health meeting of the American Phytopathological Society in Ohio gave Agdia the opportunity to communicate our findings to plant pathologists and diagnosticians. Validation data produced with Dr. Stewart Gray and Jason Ingram (USDA ARS, Cornell University) supporting utility of the assay for screening potato tubers was the focus of this presentation. Validation data supporting test use in ornamental crops was presented at the International Society for Horticultural Science IX International Symposium of New Ornamental Crops in Guadalajara, Mexico. Agdia was able to show that the test is not only sensitive and specific, but in collaboration with Dr. Hanu Pappu (WSU) we demonstrated that the test detects diverse virus isolates. This research will be published in an upcoming volume of Acta Horticulturae. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Tobacco rattle virus is a plant pathogen that infects hundreds of plant species, causing significant economic damage to ornamental and food crops (potato in particular). Diagnostics and preventive testing are key to managing this pest, however current detection methods are limited by their specificity and sensitivity. Agdia has successfully addressed these challenges at the conclusion of Phase I of this SBIR grant with the commercial release of the AmplifyRP XRT test for TRV. This new test is an essential tool for growers, researchers, and diagnostic clinics in their efforts to identify and manage TRV infection. All of the objectives addressing the development of the molecular detection assay were met, in addition to completing validations required for product sale. Notable features of the TRV AmplifyRP XRT test include: sensitivity greater than or equal to published PCR methods (100 fg/uL RNA transcript), crude extract preparation that eliminates the need for RNA purification, specific detection of all TRV isolates (including NM types), 100% diagnostic sensitivity, 100% diagnostic specificity, internal controls built into every reaction, validation of the test for direct potato tuber screening, stability for international shipment and extended shelf-life, and results in 20 minutes. This TRV assay can be used with current fluorometers, such as the Agdia AmpliFire. The second component of this research grant focused on improving current fluorometric technology to create an instrument that is more accessible to users with limited resources. To that end, in Phase I of this grant project Agdia selected and evaluated component parts required for the construction of a miniature fluorometer. With the final design, we were able to demonstrate that the instrument could maintain the constant temperature required of isothermal reactions (38 to 50degC) and discriminate between fluorescence produced by an isothermal assay and background signal (p=0.000015). This data can be recorded and presented using an inexpensive microcomputer that utilizes pre-set thresholds for diagnosing positive versus negative test results. While the data positively supports the possibility of a handheld fluorometer, more research is needed to optimize the configuration of component parts for optimal sensitivity. If successful, this instrument will enable growers and diagnosticians to utilize new molecular technologies without significant capital investment.

Publications

• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2020 Citation: B. Davenport, D. Groth-Helms, R. Li, S. Zhang, J. Ingram, H. Pappu (2020) Development of a real-time duplex isothermal assay for the detection of Tobacco rattle virus and an endogenous internal RNA control in ornamental hosts. IX International symposium on New Ornamental Crops. Guadalajara, Mexio. September 30 - October 3 2019. Acta Horticulturae volume in preparation.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: B. Davenport, D. Groth-Helms, R. Li, S. Zhang (2019) Development of a real-time duplex isothermal assay for the detection of Tobacco rattle virus and an endogenous internal RNA control. APS Plant Health 2019 Annual Meeting in Cleveland, Ohio, U.S.A. August 3 - 7 2019.

Progress 09/01/18 to 08/31/19

Outputs
Target Audience:In collaborative validations and conference presentations, we were able to communicate the novel features and superior performance of the Tobacco rattle virus recombinase polymerase assay (TRV RPA) to a large share of the target audience including researchers, educators and diagnosticians. We worked directly with leading plant virologists through the course of the assay evaluation. Dr. Hanu Pappu (WSU) shared isolates that we used to demonstrate the comprehensive specificity of the test. Dr. Stewart Gray and Jason Ingram (USDA ARS at Cornell University) provided both laboratory space and materials that were used to demonstrate the high sensitivity and practical application of the test with crude potato extracts. Interactions at the APS Plant Health meeting and ISHS International Symposium on New Ornamental Crops generated significant interest in the upcoming TRV assay, both in the practical application and the new advances in RPA design. Changes/Problems:The original optical design for the miniature fluorometer produced weaker than expected results in initial testing. Due to the critical need for the device to have a sensitivity equal to or better than devices currently on the market, the optical system was re-designed. A new sensor and filter arrangement is currently being evaluated. Feasibility analysis for the prototype miniature fluorometer will be completed in April 2020. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Performance characteristics of the validated TRV duplex isothemal assay were presented to the plant science community in two conference proceedings. An overview of the test witha particular focus on potato diagnostics was presented to plant pathologists at the annual Plant Health meeting of the American Phytopathological Society in Cleveland, Ohio. This poster communicatedthe superior performance of the test as compared to other published and commercial methods and its suitability for screening dormant potato tubers. At the 9th International Symposium of New Ornamental Crops in Guadalajara Mexico, the assay was described in detail both as an oral presentation and research paper to floriculture researchers worldwide. Highlights of the presentation were the demonstrated utility of the test in screening a diversearray of ornamental tissues and the rapid, low cost assay design. What do you plan to do during the next reporting period to accomplish the goals?The goals of this project relating to the development of a TRV isothermal assayhave been met in full. Commercial release of this test, supported by fluorometers currently available, is expected in early 2020. For the duration of Phase I of the project, the focus of research will be the evaluation of the miniature fluorometer's component systems. A new optical design will be evaluated for sensitivity to ensure equivalent performance to existing fluorometric devices. At the same time, an alternative low cost heating system will be tested to ensure consistent performance over time. Programming work will continue in support ofa user-friendly interface and data display.

Impacts
What was accomplished under these goals? IMPACT Through the course of Phase I development, Agdia has produced a highly sensitive and rapid test for the detection of Tobacco rattle virus. This pathogen infects hundreds of different plant species with significant economic consequences including corky ringspot disease of potato and unattractive spotting and decline of ornamental crops. With the newly developed test, potato growers and diagnosticians can for the first time reliably detect low concentrations of the virus from crude potato tuber or plant leaf samples in 20 minutes or less. In addition, the test includes internal controls in every reaction for enhanced quality assurance and guaranteed stability for shipping worldwide. This accomplishment is the first step towards a low-cost diagnostic test system that expands accessibility to state-of-the art diagnostics. Our primary focus for the duration of the project will be optimizing the design of an inexpensive, miniaturized fluorometer that eliminates the need for expensive benchtop equipment when visualizing results. PROGRESS Agdia has met or exceeded the research objectives outlined for the development of a novel Tobacco rattle virus molecular detection assay. Primers and probes were designed and evaluated for optimal sensitivity with a representative collection of 25 TRV isolates. The reaction was then multiplexed with an endogenous internal control and re-evaluated to ensure there was no loss of performance. To simplify and speed the test process, the test was designed to work without mixing step otherwise required for recombinase polymerase amplification (RPA) technology. Different sample preparations were evaluated, and a buffer was selected for use in preparing crude extracts. Exclusive specificity testing was conducted with related viruses and 70 different plant host species, showing no false positives and reliable internal control reaction. The test underwent multiple validations for diagnostic and analytical sensitivity and specificity. Analytical testing with crude extracts showed equal sensitivity to conventional RT-PCR and greater sensitivity than Real-time qPCR. Notably, these other methods require RNA isolation. The test also showed competency of detection in crude potato tuber samples that was equal to or greater than current laboratory methods. In a validation with multiple operators and technologies, the TRV RPA showed consistent superior performance to multiple RT-PCR, RT-qPCR, and ELISA tests. Stability testing was conducted to ensure the test can be offered as a product that can be shipped for weeks at room temperature. Commercial release of the TRV RPA is expected in early 2020, before the end of Phase I development. This test can be used with fluorometric devices currently available. Primary research objectives for the low-cost, miniaturized fluorometer include evaluations of the design of thermal, optical and data subsystems for quality and cost. To date, Agdia has optimized a heating system that can be used to incubate a reaction consistently over the desired temperature range with low power inputs easily supplied with an inexpensive lithium ion battery. Alternatives to this design with even lower cost components are being evaluated. The original design of the optical system produced sensitivity of detection that was lower than expected with resolution less than current fluorometric devices. This prompted a re-design of the system, with alternative sensor and filter component parts. Evaluation of the new array is expected in late 2019. Work has also been done to program the device for a user-friendly and accurate result display using a low-cost and wireless microcomputer. Verification of the program will be conducted once the thermal and optical subsystems are finalized. With a viable optic system, a functional prototype is expected in 2020.

Publications

• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2020 Citation: B. Davenport, D. Groth-Helms, R. Li, S. Zhang, J. Ingram, H. Pappu. (2020) Development of a real-time duplex isothermal assay for the detection of Tobacco rattle virus and an endogenous internal RNA control in ornamental hosts. IX International Symposium on New Ornamental Crops. Guadalajara, Mexico. September 30 - October 3 2019.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: B. Davenport, D. Groth-Helms, R. Li, S. Zhang (2019) Development of a real-time duplex isothermal assay for the detection of Tobacco rattle virus and an endogenous internal RNA control. APS Plant Health 2019 Annual Meeting in Cleveland, Ohio, U.S.A., August 37, 2019.