Non Technical Summary
Significance of the ProblemQuick and accurate detection of foodborne pathogens is crucial for preventing outbreaks and ensuring food safety. Current methods are either too slow (1-5 days for culturing or 1.5-3 hours for molecular tests) or require specialized equipment and expertise. We need a fast, simple, and accurate test that can be used on-site, like lateral flow test kits. However, these kits have limitations.Our company, Cellbae Inc., has developed a new rapid test kit (RTK) platform, which utilizes enzymatic amplification that increases sensitivity and accuracy. We aim to create a RTKs that detect foodborne pathogens like E. coli (EC) O157:H7 and Salmonella Enteritidis (SE) quickly and accurately. With increased sensitivity, we can reduce pre-enrichment culture from 24 to 8 hours followed by testing on the RTK (within 15 min), ensuring food safety at every stage of production, processing, anddistribution.MethodsWe will first develop a large-scale production process for our enzymatic RTKs using semi-automated equipment. This involves optimizing the production protocol, sourcing materials, and establishing quality control measures to ensure the RTKs meet the required standards. We will also set up a quality management system to ensure the RTKs are safe and effective. This includes monitoring equipment, facilities, and materials, as well as validating the manufactured products through sampling and testing. Finally, we will ensure proper packaging and labeling to maintain product integrity. All procedures and records will be documented to ensure traceability and productquality.Next, we will obtain Performance Tested Method (PTM) certification by the Association of Official Analytical Collaboration (AOAC) International. The certification process involves testing and validating the performance of our RTKs for detecting Salmonella Enteritidis and E. coli O157:H7 in various food samples. We'll test the RTKs using different sample types, such as poultry, egg, and meat products, and compare the results to established reference methods. The testing will evaluate the RTKs' accuracy, reliability, and robustness, including their ability to detect low levels of bacteria. Additionally, we'll conduct stability studies to ensure the RTKs remain effective over time. The certification process involves collaboration with AOAC and independent laboratories to ensure the highest standardsaremet.GoalsAfter developing manufacturing methods and obtaining AOAC certification, we can proceed to commercialize and market the products. With RTKs that have high sensitivity and specificity, farmers will be able to detect outbreaks much earlier, and take preventive measures to prevent animal deaths and production loss. Food processing plants can also validate their processes to be pathogen-free, and be able to ship their products out confidently, without regulatory repercussions that may lead to financial losses. Regulatory authorities conducting food consignment checks can also be more confident of screening results from the RTK, and reduce the amount of tests required for confirmatory culture testing, resulting in time and cost savings.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	3260	1100	40%
712	3270	1100	10%
712	3320	1100	50%

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

Subject Of Investigation
3320 - Meat, beef cattle; 3260 - Poultry meat; 3270 - Eggs;

Field Of Science
1100 - Bacteriology;

Goals / Objectives
Our goal is to fulfill all certification requirements by the Association of Official Analytical Collaboration (AOAC) International for our lead foodborne pathogen rapid test kits (RTKs): E. coli (EC) O157:H7 and Salmonella Enteritidis (SE), and develop large-scale manufacturing methods to scale up RTK production, to aid our product launch and marketing during commercialization.Our key objectives are:Develop large-scale manufacturing methods.Production protocol with semi-automated equipment.Sourcing and selection of suitable production materials.Optimize composition/concentration of production reagents.Set up quality control (QC) and quality assurance (QA) plan and requirements.Documentation that complies with a suitable quality management system.Meet AOAC certification requirements for SE and EC O157:H7 RTKs.Inclusivity and exclusivity studies for target and non-target bacteria species.RTK characterization and robustness tests.Method comparison with gold standards, i.e. FDA's Bacteriological Analytical Manual (BAM), Food Safety and Inspection Service's (FSIS) Microbiology Laboratory Guidebook (MLG).Validation of RTK with relevant food matrices via both in-house and independent laboratory testing.Long-term stability and reproducibility/repeatability tests.Commence verification of RTK in field settings by commercial partners potentially upon AOAC certification.Understand partners' workflow.Implementing RTKs at the appropriate stages of their workflow to achieve time and cost savings.

Project Methods
Methodology for key objectivesPerformance Tested Method (PTM) certification by the Association of Official Analytical Collaboration (AOAC) International:The process begins with an application consultation with AOAC's technical consultant to determine the aims and scope of the application: assay type, target analyte(s), matrices, market, and regulatory issues. The output is a formal Validation Study Protocols for data collection and statistical analysis with acceptable performance criteria that will be followed during the Validation Study. The study will involve characterization of the robustness of RTKs in which we will vary parameters, such as sample volume, test runtime and operating temperature, to study their effect on the test results.The relevant matrices for the Salmonella Enteritidis (SE) RTK include poultry house drag swabs, egg pool, chicken carcass rinsates, raw chicken meat parts, and ground turkey. For the E. coli (EC) O157:H7 RTK, the relevant matrices include stainless steel swabs, apple cider, lettuce rinse, raw beef cubes, and raw ground beef. The matrices can be spiked with target bacteria species; reference bacteria colony isolate can be grown followed by enumeration of viable cells by dilution plate counting on suitable agar plates, such as trypticase soy agar (with 0.6% yeast extract supplement) for SE. A low target level of < 5 CFU/sample can be spiked into the prepared sample. Both spiked and non-spiked matrices will be cultured and tested with the RTK. As comparison to our RTK, the same sample matrices will also be tested using the gold standard reference method, such as FDA BAM "Chapter 4A: Diarrheagenic Escherichia coli" and "Chapter 5: Salmonella" and FSIS MLG method 4.14 and 5C.03 for SE and EC O157:H7, respectively. 3 out of 5 matrices will be validated by Cellbae, while 2 out of 5 matrices will be validated by an independent laboratory, which will follow the Validation Study Protocol.The lot-to-lot reproducibility study can be adopted from FDA-recognized consensus standards, such as Clinical and Laboratory Standards Institute (CLSI) EP05, wherein the RTKs can be challenged with a low target sample (e.g. 3× LOD) and a negative sample (in at least duplicates for each sample). This will be conducted twice daily for 20 days (need not be consecutive) for analysis of reproducibility. This is applicable for qualitative tests with 1 operator on 1 site, and will vary with the number of operators and sites performing the study. The real-time stability study is similar to the accelerated stability study done in Phase I, whereby the RTKs are stored at the highest ambient temperature claimed (i.e. 30°C) for 2-2.2 years. At regular intervals, the RTKs will be challenged with low target sample (e.g. 3× LOD) and a negative sample (in triplicates each).Development of large-scale production protocols:We will purchase a suite of semi-automated equipment that can scale up the lab-based production of RTKs in a semi-automatic manner. This process and protocol will involve optimizing the composition of reagents, such as the coating buffer for immobilization of the capture antibodies on nitrocellulose test strip, blocking and washing buffers, and stabilizing solution for drying antibodies on test kit materials. The method for large-scale conjugation of detector antibodies to the signal-generating enzymes will be developed and optimized. Next, production protocols, such as volume and concentration of capture/detector antibodies to be printed and immobilized on test strip, as well as the temperature and duration of blocking and drying of test strip materials, will be established. In addition, sourcing and selection of suitable materials, such as nitrocellulose membrane, absorbent pads, and conjugate pads will be conducted. Lastly, miscellaneous items, such as aluminum foil pouch and silica beads that are used to maintain low humidity and shelf-life/stability of the RTKs, will be sourced and selected for the assembly of a complete kit.We will also set up the quality control/assurance requirements and technical documentation, which are needed for AOAC certification. The ISO 13485:2016 provides a framework for creating a quality management system (QMS) that ensures the quality and safety of finished products. This involves manufacturing controls to ensure that the product conforms to specification, such as being able to detect 103 CFU/mL of target within 15 min when following the Instructions for Use (IFU) provided with the RTK. This also includes monitoring and control of equipment, facilities and materials, such as regular calibration of storage and production equipment and maintaining air cleanliness and controlling particle size within facility, to ensure product conformity. To validate the manufactured products, we will establish a sampling plan, such as taking 1-5% of the products and designing quality control testing, which is related to specifications (such as ensuring a limit of detection (LOD) of 103 CFU/mL). Next, we will work out proper packaging and labeling of manufactured RTKs and accessories, so that they are delivered with integrity and conformity intact. These procedures, work instructions, and records will be documented to ensure traceability and product quality.Pilot verification of RTK in field settings by commercial partners:For the field test, we would need to understand their current production and testing workflow, and determine which critical production points would warrant an RTK testing, and which testing method could be replaced by our RTK to achieve cost and time savings.MilestonesQ1-Q2:Select suitable production materials and optimize composition/concentration of production reagents.Determine production protocol for equipment.Q2-Q3:Set up quality control/assurance requirements and technical documentation.Manufacturing of RTKs for AOAC certification studies.Real-time stability test (Q2 onwards).Q3-Q4:AOAC consultation.Prepare validation study protocol for AOAC's review, and submission of PTM application.RTK characterization, robustness test, and lot-to-lot reproducibility/repeatability test.Q4-Q6:Validate with food matrices spiked with target bacteria and compare with gold standard culture method.Independent lab validation with food matrices spiked with target bacteria.Q7-Q8:PTM review and certification.Understand partners' workflow.Pilot trial of RTKs at appropriate stages of their workflow.