A Sensitive Single-tube Immunocapture Real-Time RT-PCR for Early Detection of Plant Pathogens in Crops

Plant virus infections cause tremendous economic losses in agricultural production each year in the U.S. Potential introduction of foreign pathogens which could threaten the safety of American agriculture has accelerated in recent years because of increasing international seed and plant trade. The first line of defense against such devastating viral diseases is the early and sensitive detection of the viruses before they are introduced into the U.S, or once in the U.S. before it has become widespread. Since there is no effective method to cure plant of virus diseases, eradication of introduced virus inoculum sources through early detection may prevent the harmful pathogens from further spreading into existing and newly planted crops, and thus reducing production costs and environmental impacts of introduced viruses. It has long been a challenging task to develop timely and accurate diagnostic tests for diseases caused by viruses or virus-like agents in plants in agriculture production. Traditional methods for detecting plant pathogenic viruses include biological assay on indicator plants, serological tests such as enzyme-linked immunosorbent assay (ELISA) and immuno-strips, and electron microscopy. Biological indexing assays are time-consuming taking weeks or even months to complete. Serological tests may not provide the sensitivity that is needed to detect low concentrations of viruses in seeds and plant tissues. Modern technologies such as reverse transcription-polymerase chain reaction (RT-PCR) provide very sensitive diagnostic assays for plant virus detection. However, these molecular techniques require lab-intensive and expensive procedures of pre-sample extraction and post-PCR detection, which make it very difficult or infeasible to test large numbers of samples. By combining two widely used virus detection methods, ELISA and RT-PCR, Immunocapture Real-time RT-PCR is proposed for the detection of plant viruses in this SBIR project. The advantages of this assay system over conventional RT-PCR include eliminating pre- and post-PCR manipulations, improving sensitivity, reducing risks from contamination, and shortening the time needed for an accurate disease diagnosis. Savings on labor and materials in sample preparation significantly reduces the total cost for each test. This immunocapture Real-time RT-PCR assay will provide one of the most sensitive diagnostic tools for detection of plant viruses in many economically important crops. Sensitive, reliable and user-friendly commercial products will be developed based on this technology. It allows us to perform an entire assay in a single PCR reaction tube within a relatively short time. This cost-effective diagnostic system will be of benefit to research scientists, inspectors, growers and diagnosticians, and thus, improve crop protection, increase production efficiency and increase environmental benefits. Therefore, it will directly enhance protection and safety of the nation's agriculture and food supply, and also indirectly enhance international competitiveness of American agriculture and protect the nation's natural resource base and environment.