Progress 09/01/20 to 04/30/21
Outputs
Target Audience:•Our main target audiences were meat and poultry processing facilities. We reached out to 185 meat and poultry processing facilities over the course of the project. Our efforts involved introducing our company to the facilities, explaining our technology to the facilities, and asking the facilities what they would want from our technology and company. There were 21 facilities who were interested in hearing more about our device. Importantly, there were two local Northern California meat and poultry processing facilities who were interested in working with us directly for early-stage field testing of our product. • Additional target audiences that we discovered during the project were other agricultural-related businesses, such as wineries, farms, and more. We reached out to 101 of these types of businesses. There were 26 who were interested in hearing more about our device. Importantly, there was a Hawaiian/Korean sauce company who was interested in working with us directly for early-stage field testing of our product. • Another target audience was the Linnes laboratory at Purdue University. We previously collaborated with the Linnes laboratory for several years and wanted to continue our collaboration through this USDA SBIR Phase I grant. Therefore, we held monthly meetings with the Linnes laboratory, which resulted in a productive exchange of research findings. Additionally, we routinely communicated with the Linnes laboratory via email, text, Slack channels, etc. regarding a range of day-to-day laboratory research topics. Changes/Problems:Our plan to manufacture our own saliva collection containers turned out to be more costly and more time-consuming than desired for quickly having a product ready. As a result, we obtained inexpensive off-the-shelf containers, tested them, and showed that they could withstand leak tests and drop tests, making them suitable for our application. Also, the strand-displacement probes did not improve our assay sensitivity. However, we tested a range of additional reagent components and identified a new reagent formulation that significantly improved our assay sensitivity and brought the sensitivity into the range of other FDA-authorized saliva-based tests. Finally, our in-house lyophilization protocol yielded promising results but needed improvement. Therefore, we hired BIOLYPH to add additional excipients to our assay formulation to ameliorate any issues. Overall, the project was a success. What opportunities for training and professional development has the project provided?The project provided opportunities related to the FDA, diagnostics technology, and software: FDA. The project provided extensive professional development regarding FDA knowledge for all staff. For example, multiple OmniVis team members attended town hall meetings offered directly by the FDA where current topics regarding FDA regulatory pathways were discussed. Also, OmniVis hired an FDA consulting group called Parexel, and Parexel imparted significant knowledge to the OmniVis team regarding intricacies of the FDA EUA pathway. Our interactions with Parexel were so helpful that we submitted a pre-EUA package and are currently preparing an official EUA package based on feedback we obtained from the FDA during the pre-EUA submission process. Diagnostics technology. Multiple OmniVis team members attended a virtual retreat hosted by the Purdue University Weldon School of Biomedical Engineering focused on clinical an translational topics related to diagnostics, disease detection, digital health, wearable devices, and more. Software. We routinely use SolidWorks for designing various components of our device, and their parent company called Dassault Systemes held a virtual conference called the Biovia Conference. It focused on laboratory informatics for the life sciences field, and we acquired new knowledge about software technologies for modeling, simulation, quality, and manufacturing. How have the results been disseminated to communities of interest? We disseminated results by contacting 185 meat and poultry processing facilities and 101 other agricultural-related businesses, including wineries, farms, and more, all of whom may be future customers, testing partners, etc. When contacting these businesses, we introduced OmniVis and the technology that we were developing as part of this USDA SBIR Phase I grant. A total of 47 businesses were interested in hearing more about OmniVis, and three were specifically interested in participating in early-stage field testing. We also disseminated results by having monthly meetings with the Linnes laboratory at Purdue University. These meetings were science-focused and involved a robust exchange of scientific information between industry scientists at OmniVis and academic researchers at Purdue. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
? Objective 1 was to develop a saliva collection container for SARS-CoV-2 testing. The following accomplishments were made: The major activities completed were (1) creation of two saliva collection cup designs for in-house manufacturing, (2) a cost analysis comparing saliva collection containers manufactured in-house to those purchased off-the-shelf, and (3) testing of two different off-the-shelf saliva collection containers. The tests of the off-the-shelf containers included leak tests, drop tests, and loop-mediated isothermal amplification (LAMP) tests using saliva collected from the cups/funnels. The data is described as follows. Firstly, manufacturing our own saliva collection cup was estimated to cost thousands of dollars, while the cost of simply purchasing an equivalent amount of off-the-shelf items was $280. Leak tests and drop tests were performed, and results were graded using a scale of 1 to 3, where 1 is total leakage with the lid detaching, 2 is partial leakage with the lid remaining attached, and 3 is no leakage at all. The saliva collection cup had leak test scores of 3, 3, and 3 (n=3) and drop test scores of 3, 2, and 2 (n=3) and was capable of collecting saliva that was successfully used in LAMP reactions. The saliva collection funnel had leak test scores of _, _, and _ (n=3) and drop test scores of _, _, and _ (n=3) and was also capable of collecting saliva that was successfully used in LAMP reactions. The following summary statistics were calculated. The saliva collection cup had an overall leak test score of 3+/-0 and an overall drop test score of 2.3+/-0.6. The saliva collection funnel had an overall leak test score of _+/-_ and an overall drop test score of _+/-_. Overall, the saliva collection funnels performed better than the cups. Also, the funnels were more preferred because they could be used to collect smaller saliva samples (<5mL), while the cups were intended for larger samples (>100mL). The key outcomes of objective 1 were that a 5-mL saliva collection funnel/tube could be sucessfully used to collect saliva samples and that the collected samples are capable of being used in LAMP reactions. The off-the-shelf funnel/tube costs only $0.28 per unit, which is significantly less expensive than manufacturing our own saliva collection funnels/tubes. Objective 2 was to test whether strand-displacement probes make a SARS-CoV-2 LAMP assay more effective at detecting low sample concentrations. The following accomplishments were made: The major activities completed were (1) the designing and testing of strand-displacement (SD) probes, (2) the testing of a range of additional reagents to improve the sensitivity of our SARS-CoV-2 assay, and (3) establishment of a lyophilization protocol to freeze-dry assay reagents for eventual shipping and storage of the product at room temperature. As mentioned in our interim technical report, after testing the SD probes, we found that the probes did not improve assay sensitivity. Also, we discovered that optimal performance of SD probes requires a preliminary heating step of 95°C for 1 minute followed by 0.1°C/s cooling to room temperature. This heating step cannot be incorporated into our device without extensive re-design of the technology. For these reasons, we pivoted away from the use of SD probes for improvement of assay sensitivity. Instead, we tested a range of other assay components, resulting in multiple successes. The other reagents we tested were different DNA polymerases (NEB's Bst 3.0 and WarmStart), components to prevent spurious amplification (dUTP/UDG to prevent carryover contamination and Tte-UvrD Helicase to prevent non-specific amplification in controls), a range of different primer gene targets (NSP3, E1, N1, N2, and 10 additional primer sets designed with the NEB design tool), various saliva concentrations (0%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, and 50%), sample preparation chemicals (Tween-20, Triton X-100, Zymo Research's DNA/RNA Shield buffer), and filtering of the fluorescent beads required for our assay in order to remove excessive amounts of EDTA from the bead buffer solution. Finally, we developed a lyophilization protocol to freeze-dry our assay reagents. The protocol involved adding an excipient to the assay formulation, flash-freezing the reagents in liquid nitrogen, further freezing at -80°C, and then freeze-drying the reagents. The data is described as follows. After testing the range of reagent components mentioned in the paragraph above, we arrived at a new assay formulation that resulted in amplification of SARS-CoV-2 samples while 15% saliva and fluorescent polystyrene beads were present in the reaction mixture, which we were not able to achieve using our previous assay formulation. We found that amplification occurred 100% of the time in 105, 104, and 103 copies/reaction concentrations, approximately 50% of the time in the 102 copies/reaction concentration, and 0% of the time in the 101 copies/reaction concentration (n=5). Next, we wanted to further improve the sensitivity of the assay, so we performed additional testing. We discovered that the buffer in which the fluorescent beads were suspended contained an amount of EDTA that was excessive for LAMP reactions to the point that it may have significantly sequestered Mg2+ ions during the reaction. Therefore, we removed the bead buffer, replaced it with water, and performed the same LAMP reaction. We saw a significant improvement in the sensitivity of the assay (i.e., approximately a 100-fold improvement in sensitivity). Finally, we used our lyophilization protocol to freeze-dry a batch of reagents and tested the reagents in a test kit for their ability to undergo a LAMP reaction. The test was successful--SARS-CoV-2 samples amplified, while negative control samples did not amplify. Summary statistics were calculated as follows. The FDA defines the limit of detection (LoD) for an assay as the number of viral genome copies per uL of sample that can be detected at least 95% of the time. Before removing the bead buffer from our assay, the LoD was 217 copies per uL of input sample. After removing the bead buffer from the assay, the LoD was 2 copies per uL of input sample. When using lyophilized reagents, LAMP reaction efficiency appeared to be slightly reduced. Therefore, we started working with BIOLYPH, a lyophilization manufacturer, and BIOLYPH will add additional excipients into our assay formulation to improve reaction efficiency. The key outcomes of objective 2 were that (1) our improved assay formulation is capable of amplifying SARS-CoV-2 samples in a 15% saliva solution that contains fluorescent beads, (2) the LoD of the improved assay is 2 copies/uL, and (3) the assay reagents can be lyophilized and used to perform LAMP reactions. Impact statement: This project resulted in methods for a saliva-based SARS-CoV-2 diagnostic assay for use at meat and poultry processing facilities, including an easy-to-use saliva collection funnel and a formulation for reagents/chemicals that can be included in the diagnostic test kit.
Publications
- Type:
Websites
Status:
Published
Year Published:
2021
Citation:
https://omnivistech.medium.com/omnivis-2020-year-end-recap-3bff1de3b79e
|
Progress 09/01/20 to 04/30/21
Outputs
Target Audience: Our main target audiences were meat and poultry processing facilities. We reached out to 185meat and poultry processing facilities over the course of the project. Our efforts involved introducing our company to the facilities, explaining our technology to the facilities, and asking the facilities what they would want from our technology and company. There were 21 facilitieswho were interested in hearing more about our device.Importantly, there were twolocal Northern California meat and poultry processing facilities who were interested in working with us directlyfor early-stage field testing of our product. Additional target audiences that we discovered during the project were other agricultural-related businesses, such as wineries, farms, and more. We reached out to 101of these types of businesses. There were 26 who were interested in hearing more about our device.Importantly, there was a Hawaiian/Korean sauce companywho was interested in working with us directlyfor early-stage field testing of our product. Another target audience was the Linnes laboratory at Purdue University. We previously collaborated with the Linnes laboratory for several years and wanted to continue our collaboration through this USDASBIR Phase I grant. Therefore, we held monthly meetingswith the Linnes laboratory, which resulted in a productive exchange of research findings. Additionally, we routinely communicated with the Linnes laboratory via email, text, Slack channels, etc. regarding a range of day-to-day laboratory research topics. Changes/Problems:Our plan to manufacture our own saliva collection containers turned out to be more costly and more time-consuming than desired for quickly having a product ready. As a result, we obtained inexpensive off-the-shelf containers, tested them, and showed that they could withstand leak tests and drop tests, making them suitable for our application. Also, the strand-displacement probes did not improve our assay sensitivity. However, we tested a range of additional reagent components and identified a new reagent formulation that significantly improved our assay sensitivity and brought the sensitivity into the range of other FDA-authorized saliva-based tests. Finally, our in-house lyophilization protocol yielded promising results but needed improvement. Therefore, we hired BIOLYPH to add additional excipients to our assay formulation to ameliorate any issues. Overall, the project was a success. What opportunities for training and professional development has the project provided?The project providedopportunities related to the FDA, diagnostics technology, and software: FDA. The project provided extensive professional development regarding FDA knowledge for all staff. For example, multiple OmniVis team members attended town hall meetings offered directly by the FDA where current topics regarding FDA regulatory pathways were discussed. Also, OmniVis hired an FDA consulting group called Parexel, and Parexel imparted significant knowledge to the OmniVis team regarding intricacies of the FDA EUA pathway. Our interactions with Parexel were so helpful that we submitted a pre-EUA package and are currently preparing an official EUA package based on feedback we obtained from the FDA during the pre-EUA submission process. Diagnostics technology. Multiple OmniVis team members attended a virtual retreat hosted by the Purdue University Weldon School of Biomedical Engineering focused on clinical an translational topics related to diagnostics, disease detection, digital health, wearable devices, and more. Software. We routinely use SolidWorks for designing various components of our device, and their parent company called Dassault Systemes held a virtual conference called the Biovia Conference. It focused on laboratory informatics for the life sciences field, and we acquired new knowledge about software technologies for modeling, simulation, quality, and manufacturing. How have the results been disseminated to communities of interest? We disseminated results by contacting 185 meat and poultry processing facilities and 101 other agricultural-related businesses, including wineries, farms, and more, all of whom may be future customers, testing partners, etc. When contacting these businesses, we introduced OmniVis and the technology that we were developing as part of this USDA SBIR Phase I grant. A total of 47 businesses were interested in hearing more about OmniVis, and three were specifically interested in participating in early-stage field testing. We also disseminated results by having monthly meetings with the Linnes laboratory at Purdue University. These meetings were science-focused and involved a robust exchange of scientific information between industry scientists at OmniVis and academic researchers at Purdue. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1 was to develop a saliva collection container for SARS-CoV-2 testing. The following accomplishments were made: The major activities completed were (1) creation of two saliva collection cup designs for in-house manufacturing, (2) a cost analysis comparing saliva collection containers manufactured in-house to those purchased off-the-shelf, and (3) testing of two different off-the-shelf saliva collection containers.The tests of the off-the-shelf containers included leak tests, drop tests, and loop-mediated isothermal amplification (LAMP) tests using saliva collected from the cups/funnels. The data is described as follows. Firstly, manufacturing our own saliva collection cup was estimated to cost thousands of dollars, while the cost of simply purchasing an equivalent amount of off-the-shelf items was $280. Leak tests and drop tests were performed, and results were graded using a scale of 1 to 3, where 1 is total leakage with the lid detaching, 2 is partial leakage with the lid remaining attached, and 3 is no leakage at all. The saliva collection cup had leak test scores of 3, 3, and 3 (n=3) and drop test scores of 3, 2, and 2 (n=3) and was capable of collecting saliva that was successfully used in LAMP reactions. The saliva collection funnel had leak test scores of _, _, and _ (n=3) and drop test scores of _, _, and _ (n=3) and was also capable of collecting saliva that was successfully used in LAMP reactions. The following summary statistics were calculated. The saliva collection cup had an overall leak test score of 3+/-0 and an overall drop test score of 2.3+/-0.6. The saliva collection funnel had an overall leak test score of _+/-_ and an overall drop test score of _+/-_. Overall, the saliva collection funnels performed better than the cups. Also, the funnels were more preferred because they could be used to collect smaller saliva samples (<5mL), while the cups were intended for larger samples (>100mL). The key outcomes of objective 1 were that a 5-mL saliva collection funnel/tube could be sucessfully used to collect saliva samples and that the collected samples are capable of being used in LAMP reactions. The off-the-shelf funnel/tube costs only $0.28 per unit, which is significantly less expensive than manufacturing our own saliva collection funnels/tubes. Objective 2 was to test whether strand-displacement probes make a SARS-CoV-2 LAMP assay more effective at detecting low sample concentrations. The following accomplishments were made: The major activities completed were (1) the designing and testing of strand-displacement (SD) probes, (2) the testing of a range of additional reagents to improve the sensitivity of our SARS-CoV-2 assay, and (3) establishment of a lyophilization protocol to freeze-dry assay reagents for eventual shipping and storage of the product at room temperature. As mentioned in our interim technical report, after testing the SD probes, we found that the probes did not improve assay sensitivity. Also, we discovered that optimal performance of SD probes requires a preliminary heating step of 95°C for 1 minute followed by 0.1°C/s cooling to room temperature. This heating step cannot be incorporated into our device without extensive re-design of the technology. For these reasons, we pivoted away from the use of SD probes for improvement of assay sensitivity. Instead, we tested a range of other assay components, resulting in multiple successes. The other reagents we tested were different DNA polymerases (NEB's Bst 3.0 and WarmStart), components to prevent spurious amplification (dUTP/UDG to prevent carryover contamination and Tte-UvrD Helicase to prevent non-specific amplification in controls), a range of different primer gene targets (NSP3, E1, N1, N2, and 10 additional primer sets designed with the NEB design tool), various saliva concentrations (0%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, and 50%), sample preparation chemicals (Tween-20, Triton X-100, Zymo Research's DNA/RNA Shield buffer), and filtering of the fluorescent beads required for our assay in order to remove excessive amounts of EDTA from the bead buffer solution.Finally, we developed a lyophilization protocol to freeze-dry our assay reagents.The protocol involvedadding an excipient to the assay formulation, flash-freezing the reagents in liquid nitrogen, further freezing at -80°C, and thenfreeze-dryingthe reagents. The data is described as follows. After testing the range of reagent components mentioned in the paragraph above, we arrived at a new assay formulation that resulted in amplification of SARS-CoV-2 samples while 15% saliva and fluorescent polystyrene beads were present in the reaction mixture, which we were not able to achieve using our previous assay formulation. We found that amplification occurred 100% of the time in 105, 104, and 103 copies/reaction concentrations, approximately 50% of the time in the 102 copies/reaction concentration, and 0% of the time in the 101 copies/reaction concentration (n=5). Next, we wanted to further improve the sensitivity of the assay, so we performed additional testing.We discovered that the buffer in which the fluorescent beads were suspended containedan amount of EDTA that was excessive for LAMP reactions to the point that it may have significantly sequestered Mg2+ ions during the reaction. Therefore, we removed the bead buffer, replaced it with water, and performed the same LAMP reaction. We saw a significant improvement in the sensitivity of the assay (i.e., approximately a 100-fold improvement in sensitivity). Finally, we used our lyophilizationprotocol to freeze-dry a batch of reagents and tested the reagents in a test kit for their ability to undergo a LAMP reaction. The test was successful--SARS-CoV-2 samples amplified, while negative control samples did not amplify. Summary statistics were calculated as follows. The FDA defines the limit of detection (LoD) for an assay as the number of viral genome copies per uL of sample that can be detected at least 95% of the time. Before removing the bead buffer from our assay, the LoD was 217 copies per uL of input sample. After removing the bead buffer from the assay, the LoD was 2 copies per uL of input sample. When using lyophilizedreagents, LAMP reaction efficiency appeared to be slightly reduced. Therefore, we started working with BIOLYPH, a lyophilization manufacturer, and BIOLYPH will add additional excipients into our assay formulation to improve reaction efficiency. The key outcomes of objective 2 were that (1) our improved assay formulation is capable of amplifying SARS-CoV-2 samples in a 15% saliva solution that contains fluorescent beads, (2) the LoD of the improved assay is 2 copies/uL, and (3) the assay reagents can be lyophilized and used to perform LAMP reactions. Impact statement:This project resulted in methods for a saliva-based SARS-CoV-2 diagnostic assay for use at meat and poultry processing facilities, including an easy-to-use saliva collection funnel and a formulation for reagents/chemicals that can be included in the diagnostic test kit.
Publications
- Type:
Websites
Status:
Published
Year Published:
2021
Citation:
https://omnivistech.medium.com/omnivis-2020-year-end-recap-3bff1de3b79e
|