Recipient Organization
PATHSENSORS, INC.
800 W BALTIMORE ST STE 405
BALTIMORE,MD 21201
Performing Department
(N/A)
Non Technical Summary
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.
Animal Health Component
0%
Research Effort Categories
Basic
(N/A)
Applied
50%
Developmental
50%
Goals / Objectives
The Technical Objectives for Phase II will expand the platform through:Adding a new target pathogen: Candidatus liberibacter spp.Independent lab validation for both Ralstonia and CaLasFull commercial-quality software integration to the off-the-shelf 96-well plate readerOptimizing the sample prep methods for 96-well platesObjective 1: Validate Ralstonia Detection System: The goals of the external validation studies for the Ralstonia assay will be to confirm the limit of detection that PathSensors found internally; to determine the ability to detect the pathogen in live plant samples, including asymptomatic tissue; and to confirm that the assay does not cross-react with other bacteria that infect ornamental and solanaceous plants.Objective 2: Develop HLB Biosensor: Working in collaboration with Dr. John Hartung from the USDA ARS, we will be using antibodies created against outer membrane proteins to Candidatus Liberibacter asiaticus to create a biosensor for screening for citrus greening disease.Objective 3: Validate HLB Biosensor: The goals of the external validation studies for the HLB assay will be to confirm the limit of detection that PathSensors finds internally; to determine the ability to detect the pathogen in mite vectors and a variety of plant tissues, symptomatic or not; and to confirm that the assay does not cross-react with other bacteria and viruses that infect citrus plants.Objective 4: Sample Prep Development and Optimization: To expedite and develop a universal sample prep, we will develop a 96-well sample prep based upon bead-based tissue disruption. In addition, we will investigate vacuum-membrane based methods for samples requiring non-standard sample buffer.Objective 5: Plate Reader Customization: The test instrument for the Phase I work was the Promega GloMax 96-well plate reader. However, in the 2nd quarter of 2016, Promega expects to phase out the current model and replace it with a completely overhauled version. We have worked with Promega to secure early access to the new hardware and its associated documentation and software interface. As a result, we will have enhanced ability to customize both the computer's user interface to the device as well enhance PathSensors branding to both the hardware and interface components.
Project Methods
Objective 1, Ralstonia Independent Validation: PathSensors has agreements with two external laboratories to validate the 96-well plate Ralstonia assay. Dr. Carrie Harmon is the Director of the University of Florida Extension Plant Pathogen Diagnostic Center and an expert on the detection of Ralstonia. She has agreed to dedicate her laboratory and personnel resources to validating the high throughput CANARY® assay for Ralstonia. Dr. Harmon has agreed to perform hundreds of assays, with samples including pure Ralstonia cultures, plant material spiked with bacteria, and true infected field samples. PathSensors will provide the instrument and reagents, and Dr. Harmon will provide the lab space, personnel, and sample material. The testing will include 880 assays of pure Ralstonia cultures, including Race 3 biovar 2, and an additional 880 assays of geranium leaf cuttings spiked with pathogen to confirm the LOD that was determined in-house. The bulk of the remaining samples assayed will be of infected plant material, with tests performed at various time points post infection to evaluate the assay's ability to detect Ralstonia in the presence and absence of symptoms. This testing will incorporate geraniums and other ornamentals, as well as solanaceous plant material such as potato and tomato leaves.Dr. Qi Huang is a Research Plant Pathologist at the USDA Agricultural Research Service (ARS), where she specializes in bacterial diseases of ornamental plants, particularly Ralstonia. She has an extensive collection of Ralstonia isolates from all over the world, including serovars already present in the United States and those the USDA is especially worried about entering the country, like Race 3 biovar 2. The format and number of tests performed at Dr. Huang's lab will be similar to those of the testing at Dr. Harmon's lab, with the addition of 88 samples of an exclusivity panel of closely related bacteria. The specific bacteria will be determined jointly with Dr. Huang based on their relevance and availability.Objective 2, HLB Biosensor & Assay Development: When engineering a new biosensor, typically after a target antigen is identified, monoclonal antibodies are generated. The heavy and light chain highly variable regions (VH and VL) from the monoclonal antibodies are cloned into expression vectors already containing scaffold DNA encoding IgM-type antibody heavy and light chain constant regions. Dr. John Hartung has agreed to supply PathSensors with the sequences for the VH and VL regions from anti-Candidatus Liberibacter antibodies. The VH and VL regions for each antibody will be cloned into PSI's proprietary expression vectors and transfected into the parental biosensor cell line. The resulting clones will be screened using recombinantly expressed surface proteins specific to the antibodies used to create the biosensor. Clones that are responsive to the recombinant protein will further be screened with enumerated CaLas-infected psylid extract. Once the biosensor's sensitivity is validated, we will then screen the biosensors for reaction to sample matrix material and cross-reactivity to other known citrus pathogens. Finally, we will test the samples against infected psyllid and plant material using the universal plant extraction developed in Technical Objective 4 to confirm the detection before proceeding with validation.Objective 3, HLB Independent Validation: PathSensors has engaged several potential partners in the validation and early use of a CANARY® assay for the detection of HLB. Dr. John Hartung, who has agreed to supply PathSensors with DNA sequences for VH and VL regions of scFv antibodies, also has a large collection of CaLas bacteria and D. citri mites that he has offered to make available for testing. Once the biosensor are created, the collection of bacteria and sample matrices from which the antibodies were created makes the most sense as an initial point of assay validation. Testing against Dr. Hartung's library will prove the ability of the biosensor to detect CaLas and the ability of the CANARY® assay to eliminate nonspecific signaling from various sample matrices, including citrus leaves and D. citri.Dr. Wenbo Ma is an Associate Professor of Plant Pathology at the University of California-Riverside who specializes in bacterial citrus diseases, particularly HLB. She has an extensive collection of CaLas bacteria and associated proteins, as well as citrus plant material infected with HLB. This library will be used to test the effectiveness of the CANARY® assay at detecting HLB before, during, and after presentation of symptoms by the plant tissue.Objective 4, Sample Prep Optimization: The current sample prep protocol for the Ralstonia single tube assay involves combining 5 plant samples-cut with disposable razors-into 5 mL of sample buffer, shaking the tube, and transferring to an assay tube. While this process is fast and simple, when put in a high-throughput application it is time consuming. For this reason, we need to optimize the sample preparation methods.For this we will develop a method using stainless steel scalpels for soft plant material and a twenty-gauge needle. The samples will be excised, and the metal instruments will be sterilized with an infrared micro-sterilizer. The excised samples will be placed in a 96-deep-well plate containing assay buffer and sample lysing matrix. For each sample type, a lysing matrix will be tested to determine the optimal conditions with a FastPrep-96 instrument. Using a FastPrep-96 instrument to prepare samples will both expand the range of tissue types that are compatible with CANARY® technology and streamline sample prep for existing tissue types.In addition to using the FastPrep-96 instrument, there may be sample preps unique to the pathogen in question. For example, Dr. Hartung's laboratory has demonstrated that direct tissue blot immunoassays can detect CaLas in infected tissue. To adapt this protocol to the 96-well instrument, we will test membrane lined luminometer plates. For this testing, we will work with Dr. Hartung to obtain infected plant material and determine the LOD. Finally, should there be a need to directly assay a liquid, such as irrigation water, we will test 96-well filter plates. For this testing, we will test differing dilutions of Ralstonia to determine our LOD.Objective 5, Instrument Customization Objective:PathSensors' Customized Software Interface: PathSensors will develop a customized interface to the new plate reader hardware. This software will provide the operator of the device with a simple and easy-to-use method to operate the plate reader, removing the need for understanding the complex setup routines typically necessary to operate such a device.We have received advanced documentation from Promega on the Application Programmer's Interface (API) that will be available for this purpose, which will permit:Communicating with the plate readerSetting assay parametersRetrieving raw data from the plate readerThis API will permit us to hide the complexity of plate reader settings and operation behind a simple user interface, walking the user through the assay process and instrument operation and providing a simple positive or negative test result for each plate well.PathSensors will use the standard process of software engineering (Analysis, Requirements Specifications, Design, Implementation, Testing, Operations and Maintenance) throughout the development of the instrument interface software. This process is understood well by PathSensors; we have successfully implemented such customized data import with single-tube luminometers and bare PMT (photomultiplier tube) modules in the past. PathSensors' current commercial single sample instrument (Zephyr) has a full assay user interface that was developed by our in-house software development resources.