Progress 09/01/16 to 11/30/19
Outputs
Target Audience:Lynntech's ALARM system is designed to detect bacterial pathogens that contaminate our food supply. Therefore, the target audience for this project includes all connected with the production, packaging, and marketing of foodstuff. This group consists of farmers, those involved in agricultural transportation, processing, and distribution, as well as consumers. Changes/Problems:Throughout this project we encountered several problems. The first dealt with the huge delay in finalizing our contract. While our grant was awarded in September 2016, the contract was not finalized until May 2017. This problem was resolved with the assistance of Dr. Jodi Williams and the USDA granting Lynntech a No Cost Extension. A second problem dealt with our choice of food-borne pathogens on which to focus. Originally, we developed an assay to detect E. coli, Salmonella, and Campylobacter. However, ALARM development concentrated on leafy greens such as spinach; therefore, we slightly modified our efforts and focused solely on E. coli and Salmonella. We also abandoned a FET-based detection system and moved to lateral flow analysis. Both of these changes were included in a contract modification. We found that the InnovaPrep Concentrating Pipette was more variable than expected, with respect to the consistent concentration of large volumes. Discussions with representatives at InnovaPrep indicated that a new and current model (CP Select) addresses the elution volume problem. We will include CP Select in post-Phase II development of the ALARM system. When detecting the STM2434 and STM4057 loci within the Salmonella enterica genome, we observed a low signal on the lateral flow strips. Because of this low signal, we did not multiplex the Salmonella and E. coli cPCR reactions. Further funding will allow us to improve Salmonella detection and continue development of the Lynntech ALARM system. We were unable to consistently and specifically detect the Act1 Internal Positive Control locus on lateral flow strips,although we could detect the cPCR product on polyacrylamide gels when multiplexed with Salmonella and E. coli cells. Again, with further funding, we will improve Act1 detection. What opportunities for training and professional development has the project provided?This project provided the opportunity for me (as PI) to train and mentor Lynntech Research Assistants and Research Associates who carried out all of the experiments in the lab. This training included daily meetings to discuss results and future experiments, as well as one-on-one, hands-on training in the lab. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The successes achieved during this project: We demonstrated 5-plex convective amplification of two independent loci in the E. coli genome, two independent loci in the Salmonella genome, and an Internal Positive Control (IPC), a locus within the human Act1 gene. The convective PCR system requires NO nucleic acid purification. Bacterial whole cells can serve as cPCR templates. We successfully concentrated samples, such that they were usable in convective amplification reactions, without enrichment. We developed lateral flow system to detect cPCR products derived from E.coli and Salmonella cPCR reactions. We developed an alternative lateral flow system using labeled cPCR primers to detect amplification products. We designed and constructed a convective PCR cartridge which could successfully amplify E.coli and Salmonella loci. This cartridge will serve as an integrated module in the eventual ALARM cartridge. We developed a Base Unit breadboard system designed to carryout ALARM reactions and analysis. We performed and in-house validation of the ALARM system and demonstrated 90% robust detection of two loci within the E. coli O157:H7 genome, underscoring commercial viability of the system.
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Progress 09/01/17 to 08/31/18
Outputs
Target Audience:Lynntechparticipated in the CAT-P program with LARTA in Washington DC (early November, 2016). Our Commercialization consultant from LARTA is John Selep. Through our interactions with John, we have had several meetings with members of Western Growers Society, including a face-to-face meeting in Salinas CA, at the Forbes AgTech Summit (June 28-29, 2017). Changes/Problems:During the last reporting period, we modified our contract with the USDA to include a reduction in the number of loci to be tested (seven to five) with the removal ofCampylobacterfrom our pathogen list lateral flow detection of convective PCR products, replacing graphene detection What opportunities for training and professional development has the project provided?Training for this project involves continually mentoringResearch Assistants and Research Associates who do the day-to-day work in the lab. We meet on a daily basis to discuss the day's work. We meticulously review experiments. It is important that individuals performing these studies have a comprehensive understanding of the work.This mentoring not only includes addressing background and significance of the overall project, but also the implementing new and modified protocols, techniques, and methods. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next year +, we have several goals: Develop the lateral flow strip with detection of all loci Lyophilize convective PCR reagents in the ALine sample prep module and integrateof the sample prep module with the PCR module Determine sensitivity and specifictity of the assay Perform washes with spiked leafy green samples, concentratewashes with the InnovaPrep concentrator, and detect spiked pathogens Perform testing at the Texas A&M Food Safety facility
Impacts
What was accomplished under these goals?
We haveoptimizatedour multiplex,convective PCR assay. This assay is five-plex assay - amplifying two loci fromEscherichia coli O157, two loci fromSalmonella enterica subsp. enterica, Strain LT2, and a positice-controllocus within the human beta-actin gene. We had a discussion with our Technical Contact at the USDA, Dr. Jodi Williams, and Dr. Williams agreed with us regarding a change in the contract to reduce the complexity of the reaction from seven to five. Dr. Williams also agreed with Lynntech that lateral flow is better suited for our assay (regarding pathogen detection) than the development of a graphenesensor(as originally proposed). On October 20, 2017,Lynntech received notification that the USDA had approved these modifications to the contract. Regarding lateral flow, we are currently constructing our preliminary lateral flow strips. These strips will be capable of identifying and distinguishing pathogenic loci, as well as detecting the the beta-actin positive control. The strips will also contain a lateral-flow control, which will inform the user that the strips are functioning properly. We have initaited work with ALine, Inc. Aline has designed and constructed a sample preparation moduleand a convective PCR module. The sample prep modulemixes the sample with the amplification reagents. The modulehas been tested and is functional. We anticipate minor alterations to the design of thismodule. The PCR module is designed to accomodate the sample and subject it to convective amplification. Thismoduleis being tested presently. We have purchased a volume concentrator from InnovaPrep Inc. We are using the concentrator to prepare samples (eliminating enrichment). The device concentrates cells in a liquid volume. We have taken 3,000 cells in 50mL and concentrated that sample (within minutes) to 0.3mL. From that 0.3mL, we were able to detect pathogen amplicons using 5uL in a convective PCR reaction!
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Progress 09/01/16 to 08/31/17
Outputs
Target Audience:While Lynntech was issued this award on September 21, 2016, due to delays in contract negotiations, the contract was not finalized until May 9, 2017. It was at that time that we initiated work on this Phase II project. Nonetheless, Dr. John Mueller (PI on this award) and Dr. Brian Henning (Lynntech Vice President - Business Development) did participate in the USDA Commercialization Assistance Program (CATP), November 9-10, 2016 in Washington DC. In addition, this past month Dr. Hennings attended the Forbes AgTech Summit in Salinas, CA to discuss food safety with growers and producers vegetable products. Changes/Problems:Since submitting the grant application, Lynntech has had very limited, if any, success in developing a graphene detection system or sensor. Therefore, we will examine alternative methods of amplicon detection. During the upcoming research period, we will contact Dr. JodiWilliams to discuss potential alternatives and our path forward regarding our research project. What opportunities for training and professional development has the project provided?As the PI for this project, I have and continue to mentor the Research Assistants and Research Associates who do the day-to-day work in the lab. We have daily meetings where we discuss experiments, theories behind studies to optimize our convective reactions. In addition, I work with these folks one-on-one in the lab to teach new techniques, which are applicable to the research. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will evaluate the suitability of the InnovaPrep concentrator to eliminate culture enrichment prior to amplification of the pathogen targets. We will also continue optimizing our multiplex convective amplification reaction and characterize the reactions - specifically evaluating specificity, sensitivity, and reproducibility. We will also develop the amplification cartridge and base unit.
Impacts
What was accomplished under these goals?
As indicated, due to delays in contract negotiations, the contract was not finalized until May 9, 2017. Therefore, progress has also been delayed. We have initiated optimization of our multiplex, convective PCR assay. We are currently characterizing the assay regarding sensitivity and specificity. We have InnovaPrep Inc. under contract and will be leasing a sample concentrator. This will allow us to examine bacterial contaminants in wash water, as well as leafy greens, such as spinach and lettuce. Work on the other Tasks has not yet started. We have just contracted ALine, Inc. to develop our amplification cartridge. Their work is anticipated to start within the next month.
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