Development of a multi-sample testing platform for a rapid, facile identification of Ralstonia solanacearum

For Phase I, PathSensors, Inc. developed a proof-of-concept multi-well diagnostic device, based upon CANARY technology, capable of detecting Ralstonia solancerarum down to 65 colony-forming units (cfu) in response to the NIFA SBIR solicitation. In the Recovery Plan for Ralstonia solancearum Race 3 biovar 2, which was drafted as part of the National Plant Disease Recovery System, the USDA lists one of the "highly important" research priorities as: "explor[ing] novel diagnostic techniques to detect R3b2 quickly and at low concentrations, such as ... highly sensitive antibody-responsive cell culture systems." To continue to meet this call PathSensors, through Phase II funding, will further develop the instrumentation and assays to fully integrate the testing system into a diagnostic lab setting for high throughput sample processing.CANARY® (Cellular Analysis and Notification of Antigen Risks and Yields) is a cell-based biosensor technology that demonstrates unprecedented performance in terms of speed, sensitivity, and ease-of-use when compared with other standard detection technologies. The USDA has invested over one million dollars to develop CANARY®. The USDA CPHST Laboratory in Beltsville, MD has used the Ralstonia biosensor to achieve one of the lowest limits of detection (LoD) of any biosensor-based, or other type of assay, for the detection of Ralstonia, achieving an LoD of 3 cfu from infected potato slices. Using PathSensors' engineering and design expertise, we developed instrumentation that uses commercially available, off-the-shelf parts linked together and controlled through an integrated computer software system. Users are led through all steps of the assay via a computer software program. Positive or negative sample identification will be provided by processing the CANARY® light output signal through an automated detection algorithm.To further commercialize this product, we intend to refine both our sample preparation methods and the user interface. We will also expand the catalogue of diagnostic assays by adapting this technology to current and existing plant pathogen assays such as tests for Phytophthora spp. and Citrus leprosis virus. In addition, PathSensors will develop a new biosensor for detecting the causative agent citrus greening disease, Candidatus Liberibacter spp. Citrus greening disease or Huanglongbing (HLB) is the most significant disease to threaten citrus production worldwide. The disease is widespread in both Brazil and Florida and is currently spreading to Texas and California. Once infected, citrus orchards are usually destroyed or become unproductive within eight years. The University of Florida reported that citrus greening has cost Florida's economy over $4 billion and 8,000 jobs, with losses of up to 30-40% of the expected citrus crop. To stem the spread and increase surveillance potential, a fast, high throughput assay needs to be developed for citrus greening. The instrumentation developed during Phase I answers this need and, to this end, we have partnered with Dr. John Hartung at USDA-ARS to incorporate his antibodies into the HLB assay.To build the confidence in the developed system, PathSensors will have third party diagnostic labs test and verify the performance. Upon validation, this system will be deployed and used in survey programs, field labs, and diagnostic testing facilities. Furthermore, this system will be made available to exporters to assure their plants are pathogen-free prior to shipping. Finally, PathSensors will further adapt the multiplex system for use with other existing plant pathogen CANARY® cell lines, such as those for Phytophthora and Citrus leprosis virus.