Plant diseases caused by virus infections induce tremendous economical losses for agricultural production each year in the U.S. The increasing international seed trade brings increased potential for the introduction of foreign diseases that could threaten the safety of American agriculture. Early and accurate detection of pant viruses in infested seeds and stock plants is a crucial step to eliminate virus infections and prevent further epidemiological spread among crop plants. Current virus detection is generally performed through the use of biological indexing on indicator plants, enzyme-linked immunosorbent assay (ELISA), or reverse transcription polymerase chain reaction (RT-PCR). In general, bio-indexing is time consuming and labor intensive. Although conventional RT-PCR offers greater sensitivity, it is not cost-effective and difficult to scale-up for high throughput samples. Currently, ELISA remains to be one of the best methods for plant virus detection. A sensitive immunocapture Real-time RT-PCR assay combines the advantages from two widely used virus detection methods, ELISA and RT-PCR to achieve timely and sensitive detection of plant viruses with simple sample preparation by immunocapture technology. This technology provides numerous advantages, including improving sensitivity, reducing contamination risks, eliminating pre- and post-PCR manipulations, and shortening the time needed for large sample diagnosis. This proposal presents the development of a sensitive and early surveillance system for plant viruses in crop plants using this technology. The first objective is to determine the factors affecting the efficient immunocapture of Pepino mosaic virus (PepMV) for Real-time RT-PCR analysis of tomato seed and plant tissues. The second objective is to develop a sensitive and early surveillance system to a panel of 14 tomato viruses using immunocapture Real-time RT-PCR. Once the phase I project is successful, this same strategy could be applied to develop disease surveillance systems for plant viruses in many of the economically important vegetables, fruits, ornamentals and agronomical crops. The goal of this proposal is to develop sensitive, reliable and user-friendly commercial products for routine plant virus detection basin on immunocapture Real-time RT-PCR. This cost-effective and use-friendly detection system will greatly benefit to the growers or companies with limited sources at their diagnostic labs. This novel diagnostic technology based on the immunocapture Real-time RT-PCR will significantly improve plant disease diagnostics by accurate, rapid and cost-effective identification of causal agents in agronomic or specialty crop plants at the earliest possible time relative to manifestation of disease. Therefore, it will improve crop protection resulting in reduced production costs and increased environmental benefits, and enhance protection and safety of the nation's agriculture and food supply. OBJECTIVES: This proposal presents the development of a sensitive and early surveillance system for plant viruses in crop plants using immunocapture Real-time reverse transcription-polymerase chain reaction. Objective 1. Determine factors affecting the efficient immunocapture of Pepino mosaic virus (PepMV) for Real-time RT-PCR analysis of tomato seeds and plant tissues. Anticipated outcomes: The optimum conditions for high throughout sample preparation via immunocapture will be determined and used for Real-time RT-PCR. Antibody pre-coated PCR microtiter plates or tubes will be produced. A ready-to-use Real-time RT-PCR mixture for a specific virus (PepMV) will be developed. Objective 2. Develop a sensitive and early surveillance system to a panel of 14 plant viruses in tomato using immunocapture Real-time RT-PCR, and validate the detection sensitivity and its connectivity with viral disease infection. Anticipated outcomes: Primers, TaqManTM probes and master mixture suitable for Real-time RT-PCR to a panel of viruses in tomato (14 viruses) will be developed. The Correlation between the sensitivity of Immunocapture Real-Time RT-PCR and threshold level of viral infection will be examined and validated. The reliability and performance of this detection system will be determined by testing variety of commercial samples. Once the phase I project is successful, this same strategy could be applied to develop disease surveillance systems for plant viruses in many of the economically important vegetables, fruits, ornamentals and agronomical crops. The goal of this proposal is to develop sensitive, reliable and user-friendly commercial products for routine plant virus detection with immunocapture Real-time RT-PCR. APPROACH: Objective 1. a. Antibody coating: the factors affecting the efficiency of immobilizing the capture antibodies on microtiter plates or tubes will be determined, including antibody concentrations and coating volume, post treatments, drying, packaging and storage. The various sources of PCR tubes/wells will be evaluated for their capability to absorb antibodies under coating buffer. b. Pathogen immunocapturing: the conditions for high efficient immunocapture such as tissue types, sample dilutions, buffer compositions, and incubation time and temperature will be evaluated. We will then evaluate the potential impact of drying immunocaptured PCR microtiter plates/tubes on signals generated by Real-time RT-PCR. c. PCR mixtures: the goal is to contain as many test components as possible in one mixture for Real-time RT-PCR. This PCR mixture will include primers, TaqMan probes and possible other components. Its stability will be tested under different storage conditions, and its shelf-life will be determined for certain period of storage times. The master mixtures will be in stabilized liquid form or in lyophilized form for its convenient in storage and shipping, and user-friendly in actual applications in the assay. Objective 2. Using available GenBank sequences, we will perform computer assisted sequence analysis and design virus species-specific or genus-specific primers and probes for Real-time RT-PCR to achieve reliable and broad-spectrum detection. These designed primers and probes will be evaluated for their suitability in the immunocapture Real-time RT-PCR system. We then focus our efforts to develop a plant virus surveillance system for the panel of 14 viruses on tomato crop. This can be done in a 96-well microtiter plate /tubes pre-coated with the virus-specific antibodies. Field samples prepared in the ELISA tissue extraction buffer will be applied accordingly to the whole plate. In this initial test, the standard ELISA procedure and biological inoculation will be carried out as internal controls to evaluate the effectiveness and sensitivity of the Immunocapture Real-time RT-PCR. The sensitivity of this immunocapture Real-time RT-PCR and its connectivity with viral disease infection will be examined in a series of pilot experiments with greenhouse and field samples, in comparison with ELISA and biological inoculations. Experiments will be conducted using serial dilutions of a virus inoculum prepared from a virus infected tissue to determine at what cycle threshold (Ct) value level as determined in Real-time PCR would likely result in a new disease infection through mechanical inoculation onto a biological sensitive indicator plant (Nicotiana benthamina) and statistical analysis will be performed for correlation. Necessary adjustments to the RT-PCR reaction will be made through the biological validation. Once the technique is validated, it will be used to test tissue samples from propagating seed, nursery transplanting seedlings and greenhouse tomato plants for possible viruses through the help of the collaborating. PROGRESS: 2009/06 TO 2010/01 OUTPUTS: The purpose of this project is to develop a sensitive immunocapture Real-Time RT-PCR for early detection of plant viruses in tomato crops. The first objective is to develop efficient plant virus immunocapture protocol for real-time RT-PCR, and the second objective is to develop a sensitive and early surveillance system to a panel of 14 tomato viruses. Immunocapture sample preparation: As many as 16 different kinds of PCR tubes from nine commercial sources were evaluated for their ability in immunocapturing. The PCR tubes from AJ Roboscreen and Stratagene and Corning were selected because of their strong reaction signal and low background. The selected PCR tubes were coated with capturing antibody overnight or 4 hours. Efficient immunocapturing was achieved by incubating the antibody-immobilized PCR tubes/plates with viral samples ground in sample extract buffer and followed by washing out plant tissues from tubes with PBST buffer. The target viruses captured on the PCR tubes were analyzed by ELISA, RT-PCR and Real-time RT-PCR. The antibody-coated plates treated by a special stabilizing procedure were stable for more than 6 months. Real-time RT-PCR: Using computer-assisted sequence analysis and multiple sequence alignment of the available GenBank sequences for the 14 target viruses, the newly designed primer and TaqMan probe for each virus were first evaluated in RT-PCR and finally in real-time RT-PCR using the antibody-immobilized PCR tubes/plates. Results showed that primer sets for all 14 viruses worked well in immunocapture conventional RT-PCR. For immunocapture real-time RT-PCR, 9 out of 13 viruses (AMV, INSV, PepMV, PMMoV, PVY, TEV, TAV, ToMV and TSWV) worked in the initial experiments. Using new sets of primers and probes and through optimization of RT-PCR conditions, reactions to other three viruses (CMV, TMV and TRSV) were substantially improved. Correlation between real-time RT-PCR detection and bioassay on virus infectivity: Sensitivity study showed that the developed immunocapture real-time RT-PCR could detect the target virus in preparation with crude tissue extract diluted up to 10-5 for PepMV or 10-7 for TSWV. Studies of correlation between the assay sensitivity and virus infectivity showed that immunocapture Real-time RT-PCR had higher sensitivity than that of the bioassay through virus infectivity test upon mechanical inoculation on the indicator plants. These results pointed out that real-time RT-PCR can be reliably used to detect trace of virus concentration that is capable of inducing virus infection. Lyophilized master mixture and multiplex RT-PCR: New formulas of pre-made master mixture were developed, and shown to be stable for 45 days (up to data) after being lyophilized and stores. This enzyme-including master mixture could be very useful in development of a cost-effective and user-friendly immunocapture real-time RT-PCR diagnostic kit. In addition, multiplex immunocapture real-time RT-PCR was able to simultaneously detect two viruses in a single reaction. This will allow us to significantly reduce the cost of conducting real-time RT-PCR for each individual virus in a panel virus test for crop health screen. PARTICIPANTS: Dr. Kai-Shu Ling is the proposal Co-PD/PI. Part of the project was conducted at his laboratories at the USDA facilities located at 2700 Savannah Highway, Charleston, SC 29414. Dr. Ling is a Research Plant Pathologist working at the USDA-ARS, U.S. Vegetable Laboratory in Charleston, SC. His CRIS project entitled "Genetic and Biologically-Based Management of Vegetable Crop Diseases" is listed under USDA-Agricultural Research Service National Program 303, Plant Diseases. TARGET AUDIENCES: Target audiences include plant pathology researchers, plant diagnosticians, extension plant pathologists, private consultants, government inspectors, regulatory officers, agriculture at research institutes and diagnostic companies. PROJECT MODIFICATIONS: Not relevant to this project. IMPACT: 2009/06 TO 2010/01 Plant virus diseases cause tremendous economical losses for agricultural production each year in the U.S. Deployment of an effective management strategy for plant viral diseases depends on the rapid and accurate disease diagnosis with sensitive virus detection methods. A timely and accurate diagnosis of diseases caused by viruses or virus-like agents in plants has long been a challenging task in agriculture. Current techniques for detecting plant pathogenic viruses are either time-consuming or unreliable for the samples with low viral concentration. Therefore, the sensitive, cost-effective and user-friendly kits developed based on the immunocapture Real-time RT-PCR technology will meet this urgent demand. All the objectives set on in this SBIR Phase I proposal are accomplished and our ultimatum goal is achieved. Prototype kits for some of the tomato viruses are currently under developing based on the developed immunocapture real-time RT-PCR and will be put on market in near future. The developed commercial products are sensitive, reliable and user-friendly diagnostic kits which include all assay components and ready to use. It allows users to perform an entire assay, from viral immunocapturing, captured viral RNA amplification, to amplicon detection, in a single PCR reaction tube within a relative short time. Applications of this cost-effective diagnostic system will benefit to the research scientists, inspectors, growers, as well as diagnostic laboratories and companies. AC Diagnostic is exclusively licensed a patent entitled "Method of detecting antigenic nucleic acid-containing macromolecular entities" (US Patent #5534406). This will allow us to exclusively use the patented technology on immunocapture of microorganisms for PCR assays. We also project to file a patent application to U.S. Patent and Trademark Office for the technologies developed from this SBIR Phase I grant project. The patent protection will give AC diagnostics a competitive advantage to build a sustainable business in research, development and marketing of agricultural diagnostic products based on the technology of immunocapture real-time RT-PCR. Potential customers for the products include research plant pathologists, plant diagnosticians, extension plant pathologists, government inspectors and regulatory officers, and private consultants from research institutes and diagnostic companies. AC Diagnostics is maintaining a complete data base for our current customers who are using our products. We are also collecting data for potential customers who are using or interested in use of PCR/RT-PCR in their research and in disease diagnostics. We can easily reach out to these customers and introduce our new products to them. We will conduct a series of marketing activities which include putting the new product introduction on our web pages, advertising the product on APS Phytopathology News, Plant Management Network (PMN), exhibiting the product on professional meeting, such as Annual APS Meeting, National Plant Diagnostic Network (NPDN) Meeting and organizing technique workshops, or product demonstrations at professional meeting, such as Annual APS Meeting
