Non Technical Summary
Ubiquitous in U.S. news cycles are reports of foodborne illness due to products contaminated with foodborne pathogens includingSalmonella spp., Listeria monocytogenes, E. coliH7:O157, and evenClostridium Botulinum.The National Outbreak Reporting System (NORS), curated by the Center for Disease Control (CDC), reports that between 2010 and 2016, these four foodborne pathogens caused over 1,400 outbreaks and resulted in 139 fatalities.Most recently, an outbreak linked toE. colicontaminated Romaine lettuce from Yuma, AZ, resulted in 210 illness across 36 states with 5 fatalities. The recent outbreak in Arizona highlights the problems associated with products that do not have an appropriate "kill step" in place for leafy-greens and other ready-to-eat foods that do not require the consumer to cook them before eating.The "kill-step" is a term typically used to describe a method in the manufacturing or packing process where deadly pathogens are eradicated from the final product.Unfortunately, products cannot withstand a kill-step such as heating (e.g., leafy greens), the step is cost prohibitive (e.g., irradiation), or the step simply may not be effective enough to eradicate the pathogen (e.g., heat resistant bacterial spores).In addition, some pathogens, such asE. coliandC. botulinum, secrete deadly toxins that could be present in the food even after the bacteria has been removed.For example, Botulinum neurotoxins (BoNT) are highly toxic and as little as 1 µg/kg of body weight could be lethal.Therefore, it is critical to have a monitoring program in place to identify any products that may be contaminated.Although the Food and Drug Administration curates and publishes protocols for foodborne pathogen and toxin testing, they require trained personnel and have a long time-to-result.In addition, because the tests are not field deployable, they require samples to be sent out, which can add even more time to results.However, for some products, such as leafy greens, the shelf life does not allow for a test and hold program.Therefore, a platform is needed that may be used in the field by manufacturers or by inspectors to rapidly detect adulterated products.For this project, PathSensors, Inc. (PSI) proposes to develop a portable, field-deployable platform based upon CANARY®technology.CANARY®technology is an immuno-based biosensor technology that demonstrates unprecedented performance in terms of speed, sensitivity, and ease-of-use when compared with other standard commercial methods.The USDA recently evaluated the laboratory-based platform and determined it could efficiently and accurately identify Botulinum neurotoxin in different food matrices. PSI intends to expand upon this research by adding its expertise to develop new instrumentation and adapting the current diagnostic for on-site toxin testing.After developing the new platform, PSI will collaborate with the USDA-ARS to demonstrate the utility of the newly developed system by testing spiked samples with BoNT type A, and then will conduct performance testing in food manufacturing facilities.During the testing, PSI will be able to further iterate the development to have a fully functional field-ready prototype and diagnostic.After the diagnostic's full commercialization, systems will be deployed at food manufacturing facilities and be used by food safety auditors.The system also will be made available to the USDA FSIS for testing and validation.After developing the platform, the catalog of assays will be fully expanded to include testing for Botulinum neurotoxins types (B, E, F, and H), Shiga toxins (stx1 and stx2), and Staphylococcus enterotoxin B.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	5010	2020	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
5010 - Food;

Field Of Science
2020 - Engineering;

Goals / Objectives
The over-arching goal of this project is to develop a portable, miniaturized, integrated, and field-deployable instrumentation platform based upon CANARY® technology that can be used to successfully detect the presence of bacterial toxins in food, specifically Botulinum neurotoxin (BoNT) type A. The new CANARY®-based system will enable testers in the field to identify target pathogens more rapidly and with greater ease of use, as well as broadly improve PathSensors' diagnostic services. The goal of this project will be achieved by successful completion of the following two objectives:Objective 1: Develop a rugged, portable, and autonomous diagnostic platform capable of being used in the field to detect bacterial toxins in foodObjective 2: Demonstrate functionality of new diagnostic platform in a laboratory setting and in field environment

Project Methods
Phase 1 of this SBIR project consists primarily of the engineering efforts to develop an integrated, miniaturized, field-usable version of an existing benchtop diagnostic device (Zephyr), and its verification of performance in comparison to the original device. Phase II will incorporate new pathogen detection targets.The engineering efforts can be broken down into three broad categories or tasks: hardware (electromechanical), software, and systemic. Evaluation methods for each of these three areas are discussed in detail below.Hardware Evaluations:During the hardware component selection and testing phase of this project, a variety of evaluation methods will be used. Extremely low-level light detection modules will be investigated and compared on a performance versus price basis. To compare the light detection performances of photo multiplier tubes (PMTs), avalanche photo diodes (APDs), and Silicon Photo Multipliers (SiPMs), a light-tight test jig will be created and used in conjunction with existing calibrated low-level light sources. Two key metrics for the light detector component selection will be the dynamic range (the relative light units (RLU) must span 6 orders of magnitude) and the signal-to-noise ratio (the stable light tube RLU to background RLU ratio must be equivalent to the existing Berthold Technologies Sirius-L Luminometer used in the on-the-market CANARY® benchtop testing platform).To fulfill centrifugation requirements, motors with intelligent feedback control systems will be sourced and tested in order to verify the RPMs, torque, voltage, and amperage needs of the system. One key metric will target for a 2,000xgforce within 5 seconds of rotation, with error notifications available to the system if the proper force is not achieved.Following primary component testing, the team will review and analyze the results. A report will be generated detailing the components selected before integrating them into prototype builds.Software Evaluations:PathSensors has experience developing multiple detection platforms which employ custom software. Standard full life cycle software development processes will be used to ensure that the highest quality software is produced in the shortest time at the lowest cost. For this product, PathSensors intends to use the Spiral Life Cycle Model - initial system requirements are defined in as much detail as possible, and an initial software design is created including specifications, flowcharts, and risk assessments. An initial prototype implementation will then be created, tested, and reviewed by potential system users. An iterative process will then be repeated (hence the "Spiral" naming of the mode), in which additional features and capabilities will be added to the software until a true MVP (Minimal Viable Product) is achieved. This final system will be thoroughly evaluated and tested. Routine maintenance will be carried out on a continuing basis to prevent large-scale failures and to minimize downtime.Systemic Evaluations:Following component selection and hardware/software integration into a single automated device, the entire system will be validated for performance compared against the existing benchtop CANARY® device (Zephyr).This critical comparison testing will occur across multiple dimensions. The stable light source will be used in the initial testing, /Ffollowed by luciferase testing in order to directly compare the new device's detection capabilities to the existing benchtop model.A BoNT/A testing procedure will be developed by the team and used to ensure comparable results are obtained between the new system and existing models.These tests will be conducted using multiple operators and prototype instruments to ensure accurate and repeatable test results are obtained. This validation test will result in an internally validated system ready for initial deployment and testing in the field.Finally, the field testing will be conducted at a USDA-ARS lab and at least one commercial food provider with the prototype instruments. The USDA-ARS testing will involve spiking several food matrices (e.g. apple juice, pureed carrots, milk, etc.) with BoNT/A and comparing results to previously conducted studies with the Zephyr instrument.The ruggedness and user interface will be tested with at least one fresh juice manufacturer with un-spiked product.Data and results will again be compared against the existing larger format device, with a goal to achieve 95% or greater result performance between the two devices.