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

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.