SBIR Phase I: The early detection of HLB Disease in citrus trees utilizing a novel isothermal PCR technology to identify host small RNAs

这个小企业创新研究（SBIR）第一阶段项目的更广泛的影响/商业潜力是为黄龙病（HLB）病（又名柑橘绿化病）提供一种快速，具有成本效益和灵敏的早期检测方法，以指导管理策略并限制其传播。HLB是危害全球柑橘产业的最具影响力和破坏性的病害。 目前的诊断工具要么是早期检测不可靠，要么是不可行的规模;他们是由耗时的样品制备，延迟结果报告，和高运营成本的阻碍。缺乏在早期阶段检测HLB致病细菌的存在并以最少的采样诊断HLB的技术导致疾病控制不足。其后果包括柑橘种植面积减少60%，加工用橙子减少72%，柑橘种植费用增加三倍，美国每年的成本为110 B美元。该技术将为柑橘类作物HLB的快速检测和精确防御管理提供一种新的诊断工具。 SBIR第一阶段项目将满足对HLB高度敏感的早期检测检测的迫切需求，HLB是柑橘产业的主要威胁，以支持有效的疾病管理。目前，HLB主要通过PCR检测病原体本身来检测。该测定通常是无效的，因为在柑橘树内的HLB相关细菌的滴度和分布、感染的组织类型和疾病进展的程度是高度可变的。开发新的诊断工具以在症状发展之前检测感染并且独立于直接病原体的存在是至关重要的。该项目将结合联合收割机两项创新，即与宿主树而不是病原体相关的小的非编码RNA生物标志物，以及一种新的RNA扩增方法，以开发一种在一个步骤中进行的30分钟测定，具有在接种后10周检测引起HL B的细菌感染的能力，特异性和灵敏度超过80%。第一阶段项目将1）证明使用一种新的扩增技术检测受感染树木样本中的RNA生物标志物组的可行性; 2）验证检测性能; 3）将该检测方法与传统方法进行比较。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase I project is to provide a quick, cost-effective, and sensitive early detection assay for Huanglongbing (HLB) disease (aka citrus greening disease) to guide management strategies and limit its spread. HLB is the most impactful and devastating disease plaguing the global citrus industry. Current diagnostic tools are either unreliable for early detection or infeasible to scale; they are hindered by time-consuming sample preparation, delayed result reporting, and high operation costs. The lack of technology to detect the presence of the HLB disease-causing bacterium at an early stage and diagnose HLB with minimal sampling has contributed to inadequate disease control. Consequences include a 60% decline in citriculture acreage and a 72% reduction in oranges for processing, a threefold increase in citrus farming expenses, and costs in the US of $11 B annually. This technology will provide a new diagnostic tool for rapid detection and precise, defensive management of citrus crops with respect to HLB. This SBIR Phase I project will address the critical need for a highly sensitive early-detection assay for HLB, a major threat to the citrus industry, to support effective disease management. Currently, HLB is primarily detected by a PCR assay for the pathogen itself. The assay is frequently ineffective, as the titer and distribution of HLB-associated bacteria within citrus trees, the types of tissue infected, and degree of disease progression are highly variable. Development of new diagnostic tools to detect infection prior to symptom development and independent of direct pathogen presence is crucial. This project will combine two innovations, small non-coding RNA biomarkers associated with the host tree instead of the pathogen, and a novel RNA amplification methodology, to develop a 30-minute assay performed in a single step, with the capability of detecting infection by HLB-causing bacteria at 10 weeks post-inoculation with over 80% specificity and sensitivity. The Phase I project will 1) demonstrate feasibility of using a novel amplification technology to detect an RNA biomarker panel in infected tree samples; 2) validate assay performance, and 3) compare the assay to traditional methods.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.