This Small Business Innovation Research Phase I project proposes to demonstrate the feasibility of a novel surveillance system based on real-time polymerase chain reaction (rtPCR) technologies for Citrus Huanglongbing (HLB) bacterium and Candidatus Liberibacter species. Early and accurate detection of HLB pathogens in infected asymptomatic citrus plants is a crucial step for implementing timely measures to eliminate bacterial infections and prevent further epidemic spread among crops. Current detection methods such as conventional PCR may not provide the sensitivity needed for asymptomatic citrus samples. Nested-PCR is costly and time-consuming, and more importantly, has a high risk of cross-contamination. In this proposal, we propose to develop a novel surveillance system, a single-tube nested real-time PCR for ultrasensitive detection of HLB Ca. Liberibacter asiaticus in the citrus samples with extremely low bacterial titer. With the proposed technology, the PCR assay procedure is greatly simplified, and the cost of processing each sample is significantly reduced. This makes it possible to process large numbers of samples in a relative short time period in a high through-put diagnostic system. This system will be designed to provide a useful and accurate diagnostic tool for the management and prevention of HLB diseases. The broader impact/commercial potential of this project is to provide a reliable diagnostic system to meet the challenges and demands for sensitive and cost-effective detection of HLB, currently the most devastating disease of citrus. The final product of the surveillance system is a single-tube nested-PCR test kit for sensitive detection of HLB pathogen. The HLB diagnostic kit will include all assay components in a ready-to-use format, which allows an entire PCR assay, from target RNA amplification to amplicon detection, to be performed in a single reaction tube. A prototype assay system will be developed in Phase I, and will be optimized and validated with field samples in Phase II. The goal of this proposal is to develop the HLB surveillance system with sensitive, reliable and user-friendly commercial products for HLB detection and monitoring. This HLB assay system will be useful in national citrus certification programs, plant diagnostic network systems, pathogen elimination programs, state and federal plant quarantine programs, as well as routine application in research and diagnostic laboratories
