SBIR Phase I: Novel Real-Time PCR-based Surveillance Systems for Candidatus Liberibacter Species, Bacteria Associated with Citrus Huanglongbing

本小型企业创新研究一期项目旨在验证基于实时聚合酶链反应（rtPCR）技术的新型柑橘黄龙冰（HLB）细菌和Liberibacter候选菌监测系统的可行性。在柑橘无症状感染植株中早期准确检测HLB病原菌，是及时采取措施消除细菌感染、防止疫情在作物间进一步传播的关键一步。目前的检测方法，如传统的PCR可能不能提供所需的灵敏度无症状柑橘样品。巢式pcr成本高、耗时长，更重要的是交叉污染风险高。在本研究中，我们建议开发一种新的监测系统，即单管巢式实时PCR，用于在极低细菌滴度的柑橘样品中超灵敏地检测HLB亚洲释放Ca. liberibacterium asiaticus。采用所提出的技术，PCR分析程序大大简化，处理每个样品的成本大大降低。这使得在相对较短的时间内在高通量诊断系统中处理大量样品成为可能。该系统旨在为管理和预防HLB疾病提供有用和准确的诊断工具。该项目的更广泛的影响/商业潜力是提供一个可靠的诊断系统，以满足对HLB（目前柑橘最具破坏性的疾病）敏感和经济有效检测的挑战和需求。监测系统的最终产品是用于敏感检测HLB病原体的单管巢式pcr检测试剂盒。HLB诊断试剂盒将包括即用型的所有分析成分，允许整个PCR分析，从靶RNA扩增到扩增子检测，在单个反应管中进行。第一阶段将开发一个原型分析系统，第二阶段将对现场样品进行优化和验证。本提案的目标是开发具有敏感，可靠和用户友好的商用产品的HLB监测系统，用于HLB检测和监测。该HLB检测系统可用于国家柑橘认证计划、植物诊断网络系统、病原体消除计划、州和联邦植物检疫计划以及研究和诊断实验室的常规应用。

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.