Progress 09/01/18 to 08/31/22
Outputs Target Audience:Target audienceconsists of food companies, public health officials, etc. In particular, target audience includes food production/processing companies both domestic and international. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
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?
In this successful Phase II SBIR research project, we achieved the overall goal of developing, building, and testing a novel portable prototype instrument capable of rapid detection of foodborne pathogens during pre- and post-harvest processing and distribution. Specifically, we applied our novel technology to two well established laboratory-based pathogen separation and detection methods: immunomagnetic separation (IMS) and quantitative polymerase chain reaction (qPCR). By integrating IMS and qPCR and including sample loading into a portable format, the Phase II prototype is capable of detecting as few as 1 cfu/25g of food with an enrichment time of 5 hours and a combined enrichment and analysis "end-to-end" time of 6 hours. In addition, Phase II prototype is capable performing rapid foodborne pathogen detections with sensitivity and specificity that are currently only achievable with laboratory-based manually performed procedures.
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Progress 09/01/20 to 08/31/21
Outputs Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
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?In the next reporting period, we will perform alpha testing at our facility using our Phase II prototype. This testing will consist of performing IMS on different sample matrices followed by thermal lysis using the two modules of the Phase II prototype. Sample matrices will include milk samples and meat enrichment samples. We will compare the automated Phase I prototype to manually performed procedures with the objective of achieving equivalent performance. Adjustments to the programs for IMS separation will be made as necessary depending on the results of this analysis.
Impacts What was accomplished under these goals?
In the previous reporting period, we completed Objectives 1 and 2 by finalizing our assay development, hardware development, and electronics design for our Phase II foodborne pathogen detection prototype. In the current reporting period, we have completed Objective 3 by integrating the hardware and electronics into the final Phase II prototype which consists of two modules. The first module performs immunomagnetic separation (IMS) on liquid samples to capture and concentrate foodborne pathogens. The second module performs thermal lysis to release genomic DNA from foodborne pathogens which is then eluted away from the magnetic beads for further analysis. Both modules can process up to eight 10 mL samples in parallel and include touch screen control with integrated software for user friendly operation. Below we describe in greater detail the work performed in the current reporting period for the completion of Objective 2: We completed development and testing of the PCB boards for integrated electronic control of the IMS and the thermal lysis modules. For the IMS module, The PCB board controls 1) the stepper motor driver for the magnetic actuation module, 2) the pump driver with variable voltage and bi-directional control for fluidic transport, 3) the variable voltage supply with a transistor for bead resuspension using an eccentric motor, and 4) the temperature controller. For the thermal lysis module, the PCB board controls the stepper motor for the magnetic actuation module and temperature control for thermal lysis. We have successfully integrated the PCB boards with the hardware and performed testing to ensure proper performance of all fluidic, magnetic, and temperature control operations. We also completed development of the software for control of the Phase II prototype. The software for the cell concentration module and for the cell lysis module provides the user with an interactive method of controlling and monitoring the modules during runtime. The software produces a user interface which is displayed on the touchscreen of each module. The user interface includes buttons which allow the user to tune parameters at the start of each procedure. For the cell lysis module, the user can select the lysis temperature and incubation time. For the cell concentration module, the user chooses which channels to run and how many wash cycles to perform. While the modules are active, the interface displays the current progress as well as any instructions the user needs to complete during the procedure. The user interface is implemented using web technologies such as HTML, CSS, and JavaScript. These tools are used to create the graphics which are displayed on the module's touchscreen. The user interface communicates with backend code written in the Python programming language which is responsible for controlling the module hardware. For the cell lysis module, the Python code maintains the temperature of the sample during the lysis phase and engages the magnet during the bead capture phase. For the cell concentration module, the Python code actuates the various pumps and engages the magnets.
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Progress 09/01/19 to 08/31/20
Outputs Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
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?In the next reporting period, we will perform the following: Objective 1: We will perform automated IMS and qPCR for additional foodborne pathogens. Objective 2: We will design and test a sterilization method for the automated IMS instrument to minimize sample cross contamination. We will also finalize our project model and integrate all three modules into one platform. Objective 3: We will design, test, and integrate a PCB into our final Phase II prototype. We will also finalize our software and design a friendly graphic user interface. Objective 4: We will perform alpha testing at our facility using our Phase II prototype.
Impacts What was accomplished under these goals?
During this reporting period, we have been put behind schedule due to shelter-in-place mandates administered by Alameda County in response to the COVID-19 pandemic. During these mandates, we were unable to resume normal laboratory work. Nevertheless, we still made progress with our Phase II prototype hardware development. This progress has improved our total assay sensitivity and increased the hands-free capability of our instrument. By improving the hardware for bead resuspension and fluidic transfers, we have made significant progress towards the completion of Objective 1. Additionally, we have built and tested new hardware for automated thermal lysis and magnetic actuation. As a result, we have made significant progress toward the completion of Objective 2. Below we describe in greater detail the progress made towards the completion of Objectives 1 and 2: Objective 1: We have developed two new hardware manipulations that improve our overall assay sensitivity by maximizing the washing of IMS beads to remove PCR inhibitors. We have designed and developed a new module that improves bead resuspension by utilizing a vibration platform controlled by an eccentric motor. We have also improved our fluidic pumping precision, which has allowed us to fully remove enrichment media and wash buffer during purification steps. These updates have significantly improved bead resuspension during wash steps and have minimized PCR inhibitor contamination. Objective 2: We have integrated a new heating block into our 8-sample IMS instrument to perform automated thermal lysis. We have also designed a ventilation system which allows us to control the amount of sample evaporation. This results in a more concentrated final sample, which lowers our assay's limit-of-detection. We have developed a magnetic actuation module that enables automated magnetic bead capture. This module can linearly translate along the side of the sample tubes, enabling optimal bead capture at the various volumes required during the IMS procedure. The module is also able to drop below the vibration platform, which minimizes the magnetic field on the sample during resuspension, improving washing efficiency.
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Progress 09/01/18 to 08/31/19
Outputs Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
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?In the next reporting period, we will perform the following: Objective 1: We will complete Objective 1 by performing IMS and qPCR for additional foodborne pathogens. We will work with our advisors Luisa Cheng and Vivian Wu of the USDA Western Regional Research Center to identify additional targets of interest and any attenuated strains that we may be available. Objective 2: We will complete Objective 2 by testing our 8-channel module for large volume IMS incubation and bead washing with the objective of achieving equivalent performance to the manually performed procedures. We also will complete the assembly and testing of our module for qPCR analysis of 8 samples. Objective 3: We will complete Objective 3 by integrating the modules into a single unit for construction of our final Phase II prototype. This entails miniaturization and integration of supporting instrumentation including the optical module, pneumatic actuators, reaction chambers, thermoelectric cooler (TEC), magnets and electronics with software. Objective 4: We will complete Objective 4 by performing alpha testing at our facility. In addition, we will work with our advisors Luisa Cheng and Vivian Wu to plan and initiate beta testing at the USDA Western Regional Research Center.
Impacts What was accomplished under these goals?
During the current reporting period, we have made significant improvements to our automated IMS (immunomagnetic separation) and qPCR detection methodology for rapid detection of foodborne pathogens in terms of both sensitivity and processing speed. These improvements reduced the enrichment time required to reliably detect 1 cfu of E. coli O157 on 25 g beef from 5 hours to 4 hours. By further optimizing and expanding the capabilities of the Phase I automation procedures for IMS, we have achieved significant progress toward completion of Objective 1. In addition, we have built and tested automation modules capable of performing the improved IMS procedures. As such, we have made significant progress toward the completion of Objective 2. Below we describe in greater detail the progress made toward the completion of Objectives 1 and 2: Objective 1: We have significantly improved the sensitivity of our qPCR assay by switching from an intercalating dye qPCR assay to a TaqMan qPCR assay with lower background signal. We are now able to perform up to 50 PCR cycles without any detectable amplification in the no-template-controls. This new assay improves the overall sensitivity while reducing the risk of false positives. We developed a method to increase the IMS enrichment volume from 1 mL to 10 mL without increasing the number of immunomagnetic beads that are required. We found that we can achieve similar capture efficiency, and we are therefore able to capture a much larger number of cells from the enrichment broth with the larger sample volume. The above improvements reduced the overall enrichment time required for reliable detection of 1 cfu of E. coli O157 on 25 g beef from 5 hours to 4 hours. The current overall time required for completion of the test is approximately 5 hours. Objective 2: We developed an automation module for processing of 10 mL samples with magnetic beads for immunomagnetic separation. This system performs the following steps: 1) 15 minute incubation of IMS beads with enrichment sample, 2) magnetic pull down of IMS beads, 3) partial removal of enrichment broth, 4) mechanical resuspension of the beads 4) Transfer of beads to output tubes, and 5) Washing of beads to remove PCR inhibitors. We have tested the functionality of a single sample system for isolation of E. coli O157 from enrichment broth with equivalent performance to the manually performed procedures. We have also built a module that processes 8 samples in parallel, and we are currently testing its functionality. We tested an automation module that we previously designed and constructed for thermal lysis of bacteria and elution of genomic DNA for 8 samples in parallel. For this testing, we performed thermal lysis of E. coli O157 captured on IMS beads, and elution away from the IMS beads to output PCR tubes. We tested the output of this module with our TaqMan qPCR assay and found equivalent performance compared to the manually performed procedures. It should be noted that we have successfully exceeded some of the objectives of our original Phase II proposal: In our original Phase II proposal, we targeted a combined enrichment and analysis time of 6 hours for E. coli O157. We have successfully reduced this to 5 hours. In our original proposal, our Phase II prototype design was capable of processing 3 samples in parallel. We are currently designing and building all modules such that they are capable of processing 8 samples in parallel.
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