Progress 05/07/19 to 05/06/23
Outputs
Target Audience:We have engaged multiple target audiences throughout the duration of this grant as detailed below. We have published two scientific manuscripts reporting the development of our SHERLOCK diagnostic assays for the rapid detection of White Spot Syndrome Virus and Taura Syndrome Virus in the journals Scientific Reports and Applied and Environmental Microbiology. Both articles were published as open access to ensure they are freely available to the scientific and non-scientific communities. The results of this project were presented at two aquaculture conferences: In 2020 Dr. Tim Sullivan (original PD) presented a talk entitled "CRISPR-based diagnostics for rapid detection of white spot syndrome virus in shrimp" at the Aquaculture America meeting held in Honolulu, HI. This meeting featured a broad audience of academic and government researchers, NGO members, and aquaculture professionals from the for-profit sector. This presentation was part of the session on Shrimp Health chaired by Dr. Arun Dhar (co-PD on this project) and was attended by a diverse group of US and international attendees. Co-PD Dr. Dhar and Research Scientist Dr. Shelly Wanamaker participated in the Aquaculture 2022 Triennial conference in San Diego, CA where they presented the results of this project to the aquaculture community. Dr. Wanamaker presented a talk "CRISPR-based diagnostics for disease detection in shrimp" as part of the Shrimp Health and Disease session co-chaired by Dr. Dhar to a broad audience of participants. The Triennial is the largest international aquaculture conference and tradeshow with nearly 3,000 attendees from over 90 countries. The Triennial combines the annual meetings of the Fish Culture Section of the American Fisheries Society, World Aquaculture Society, National Shellfisheries Association, and the National Aquaculture Association. We engaged with the Global Seafood Alliance, an international nongovernmental organization dedicated to advancing responsible seafood practices through education, advocacy and third-party assurances. This resulted in an invitation for Dr. Bodnar (current PD) and Dr. Wanamaker to participate in the Shrimp Summit to be held in Vietnam in July 2023. The 2023 Shrimp Summit will convene the global seafood value chain to address the critical challenges facing shrimp-farming in Asia and across the globe. In addition to our virtual participation in this summit, our latest publication will be posted on the "Resources" page to make it widely available to all summit participants. In addition to these activities for our target audience, we engaged a broad audience of students, researchers, and the general public through in-person and virtual presentations, social media platforms, and newsletter articles as detailedin the section on dissemination of results to communities of interest. Changes/Problems:The impact of the COVID-19 pandemic resulted in significant delays in experimental progress and the submission of a "Change of Objectives" request which was approved in February 2022. Experimental progress was further delayed by unforeseen technical challenges associated with a key enzyme needed for the CRISPR SHERLOCK assays. The solution required a complete redesign of the diagnostic assays for both WSSV and TSV, to use loop-mediated amplification (LAMP) and a Cas12b detection system. The Cas12b enzyme is not commercially available and therefore was custom ordered from GenScript®, arriving during the first month of our first no-cost extension period. During the no-cost extension periods we did not encounter any significant challenges and have successfully designed, optimized, and validated the second-generation SHERLOCK assays for both WSSV and TSV. We have also demonstrated the feasibility of converting these assays to a field-deployable format and identified a platform for advancing the development of these diagnostic tests through industry collaboration. What opportunities for training and professional development has the project provided?This project has provided training activities and professional development for 5 Research Scientists, 2 Research Associates, and 1 Postdoctoral fellow. The original PD on this project, Dr. Tim Sullivan, left GMGI in June 2020 to join the USDA as a National Program Leader of Animal Production Systems. This provided an opportunity for Dr. Andrea Bodnar (GMGI's Science Director) to assume leadership of this project. Under the mentorship of the PD Bodnar, Research Scientist Dr. Shelly Wanamaker joined GMGI in March 2021 and has led this project through its completion. Under the mentorship of Tim Sullivan, and then Andrea Bodnar and Shelly Wanamaker, this project provided training opportunities for two Research Associates (Veronica Pereira and Samuel Major) who were fully trained in the design and development of all aspects of the SHERLOCK assays. Both developed excellent molecular and analytical skills and generated valuable data to advance this project. Samuel Major participated in the preparation of the Applied and Environmental Microbiology manuscript and his significant contribution to this project was recognized as the lead author. GMGI Research Scientist, Matthew Harke, joined the project in 2020 to gain skills in the SHERLOCK technology and also contributed to the Applied and Environmental Microbiology manuscript as a co-author. Under the mentorship of co-PD Dr. Arun Dhar at the University of Arizona, this project provided training for a post-doctoral scientist, Dr. Roberto Cruz-Flores, who conducted the TSV challenge experiments and evaluation of viral infection using histology and RT-qPCR. Dr. Cruz-Flores participated in the preparation of both publications that were generated from this project as a co-author. He is now a Principal Scientist at the Center for Scientific Research and Higher Education (CICESE) in Ensenada, Mexico In 2020, Dr. Sullivan and Dr. Dhar attended the Aquaculture America conference held in Honolulu, HI, and presented this work as part of the Shrimp Health session chaired by Dr. Dhar. As part of this conference, Dr. Sullivan also attended a workshop on Fish Health Management. Dr. Wanamaker presented this project at the international Triennial Aquaculture Conference in San Diego in 2022, in a session chaired by the Dr. Dhar. In addition to the opportunity to present at these scientific conferences, this project has provided the opportunity for the participants to present to a broad range of audiences including undergraduate students and the general public as noted in the next section of this report. Dr. Wanamaker has gained experience forming and managing partnerships with industry including New England Biolabs, a biotech company who are experts in LAMP and Cas reagents, and Sherlock Biosciences, a biotechnology company founded in 2019 to use the SHERLOCK platform to revolutionize molecular diagnostics. How have the results been disseminated to communities of interest?The results have been disseminated through two publications and two presentations at Aquaculture conferences in addition to a variety of other venues and audiences. Through these activities we have been able to reach a broad audience including aquaculture professionals and researchers (Aquaculture America conference 2020, Triennial Aquaculture conference 2022, NOAA, Bigelow Laboratory for Ocean Sciences), undergraduate, graduate, and high school students (Northeastern University, Salem State University, Gloucester High School), and the general public (Darwin Festival, Science on the Harbor). Dr. Wanamaker presented guest lectures "Genomic tools for investigating ocean and human health" for the Ecological and Evolutionary Genomics course (EEMB 1106) at Northeastern University in Boston, MA in the Spring of 2022 and 2023. Dr. Wanamaker presented a seminar "Harnessing the power of genomics technologies for investigating marine animal health" at Northeastern University Marine Science Center Seminar Series in October 2021, and a seminar "Genomic tools for investigating marine animal health" at Bigelow Laboratory for Ocean Sciences Seminar Series, in East Boothbay, ME, in April 2022. In October 2022, Dr. Wanamaker presented the shrimp diagnostic technology as part of a seminar at the Epigenetic in Marine Biology international conference hosted at the Marine Biological Laboratory in Woods Hole, MA. As the only rapid molecular tool presented, this received a lot of interest from international researchers across governmental, non-profit, and academic entities for applications in monitoring disease and health biomarkers. Dr. Wanamaker presented a lecture "Investigating changing environments with genomics" for the AP Environmental Science class at Gloucester High School in May 2023. Dr. Bodnar presented an invited lecture "Enabling a New Era of Ocean Discovery and Understanding" for NOAA Northwest Fisheries Science Center in February 2022. Dr. Bodnar presented an invited lecture "Enabling a New Era of Ocean Exploration and Discovery with Marine Genomics" to students and the general public as part of the Darwin Festival at Salem State University in February 2022. Dr. Sullivan and Dr. Bodnar presented a virtual public lecture as part of GMGI's "Science on the Harbor" lecture series in April 2020. In addition, we engaged a broad audience through GMGI's newsletter and social media platforms which reach a wide network of followers and supporters, including hundreds of individuals around the greater New England area as well as others throughout the US. Our first publication was highlighted in the March 2020 edition of GMGI's newsletter, and our second publication was the subject of a video interview in 2023 featuring GMGI Research Associate and co-author Samuel Major. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact: The occurrence of highly virulent diseases is one of the most significant impediments to the profitability and success of aquaculture production. For shrimp farming alone, pathogens such as White Spot Syndrome Virus (WSSV) and Taura Syndrome Virus (TSV) have cost global producers billions of dollars since their detection in the 1990s. The large-scale impact of disease on production makes rapid and accurate diagnosis of infection critically important for disease management. For this reason, our goal was to develop new molecular diagnostic tools for rapid, sensitive, cost-effective tests that can be conducted in the field without sophisticated equipment or specialized training. This approach will empower farmers with the ability to rapidly respond to disease outbreaks, mitigating spread and minimizing losses without dependence on dedicated laboratory facilities. During this project, we have designed cutting-edge diagnostic assays that use a CRISPR-based molecular approach to enable detection of WSSV (a DNA virus) and TSV (an RNA virus). We demonstrated that these tests are highly specific, rapid (<1 hour), and possess comparable sensitivity to currently accepted diagnostic tests. Both the WSSV and TSV CRISPR-based diagnostic tests have been designed with fluorescence readout for quantitative results conducted in a laboratory, and we converted the WSSV assay to a paper strip colorimetric readout to demonstrate the feasibility of adapting this technology for field-based testing. Further, we optimized a field extraction method that extracts DNA/RNA in a few minutes using filter paper strips. In total, this allowed for a diagnostic process that can proceed from shrimp tissue to a positive or negative diagnosis in <1 hour with no electricity when using SHERLOCKv1 technology (RPA + Cas13a). This provides a rapid, cost-effective approach to increase disease surveillance by aquaculture producers for proactive disease management resulting in increased sustainability and profitability of production across the US and globally. Objective 1 - To design CRISPR assays for the detection of WSSV and TSV strains in laboratory settings. We designed and validated a first-generation SHERLOCK assay for WSSV and second-generation SHERLOCK assays for WSSV and TSV with high specificity, accuracy, and similar sensitivity to the accepted diagnostic methodologies of RT-qPCR and histology. The first-generation assay used recombinase polymerase amplification (RPA) prior to the CRISPR/Cas13a detection step; however, successfully adapting this assay to TSV (an RNA virus) was hindered by certain batches of the RPA polymerase that contained contaminating RNases, which resulted in reduced target detection as well as high background cleavage of the RNA reporter used by the Cas13a detection system. To solve this problem, second-generation assays were developed using loop-mediated isothermal amplification (LAMP) instead of RPA, and Cas13a was replaced with Cas12b which uses a DNA-based detection system. Fifteen different sets of LAMP primers were designed to target four different gene regions in the WSSV genome and two different gene regions in the TSV genome. LAMP primer sets were screened for speed and sensitivity using synthetic viral target, and top performing sets were selected for testing with Cas12b. For WSSV and TSV, more than 20 different guide RNAs were tested in combination with top performing LAMP primer sets for specificity, and reaction components were systematically optimized for highest sensitivity and reproducibility using fluorescence readout. The best performing primer and guide RNA combinations were used to generate standard curves using known amounts of synthetic viral target and demonstrated a reliable detection level of 200 viral copies per reaction (10 viral copies per microliter) for TSV and 100 viral copies per reaction (5 viral copies per microliter) for WSSV. These levels are comparable to those achieved with standard OIE approved molecular techniques (qPCR and RT-qPCR) marking the successful completion of Objective 1. Objective 2 - Validate these assays against the standard molecular techniques for veterinary diagnostics (qPCR and RT-qPCR). Viral challenge experiments for WSSV and TSV using P. vannamei were conducted to generate samples for validation of the SHERLOCK assays. Infection status was measured with H&E histology and viral quantification was conducted using the OIE recommended methodologies (qPCR and RT-qPCR). The DNA and RNA samples from infected shrimp used for qPCR (WSSV) and RT-qPCR (TSV) were tested using the SHERLOCK assays which revealed a strong correlation of SHERLOCK-predicted viral copy number and qPCR. We next demonstrated that the WSSV and TSV SHERLOCK assays were highly specific for their respective virus and did not give false positive results with specific pathogen-free shrimp or with shrimp infected with other common pathogens including Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV), Infectious Myonecrosis Virus (IMNV), Enterocytozoon hepatopenaei (EHP), and Vibrio parahaemolyticus which causes Acute Hepatopancreatic Necrosis Diseases (AHPND). Additionally, WSSV infected shrimp were tested in the TSV SHERLOCK assay and TSV infected shrimp were tested in the WSSV SHERLOCK assay with no cross-reactivity between these two assays. These results represent successful completion of Objective 2. Objective 3 - The impacts of the COVID-19 pandemic resulted in significant delays in experimental progress and the submission of a "Change of Objectives" request which was approved by NIFA in February 2022. As a result, the third objective was changed to: Adapt the SHERLOCK assays to lateral flow strips as an initial step to developing a field-deployable format. Our first-generation WSSV SHERLOCK assay was successfully converted from a fluorescent assay to a colorimetric lateral flow format by changing the reporter molecule, demonstrating the feasibility of this approach. This colorimetric test could detect as low as 10 copies of WSSV without cross reaction to other known shrimp viruses and with no false positive signals from pathogen-free shrimp. In order for this to be executed in a field setting without access to a lab, we optimized a simple filter paper-based nucleic acid preparation that was compatible with the lateral flow assay. The result was a field-compatible workflow that was able to extract DNA, run the SHERLOCK reaction, and generate a strip-based color readout in 60 minutes at room temperature with no lab equipment or electricity required. In the final year of the project, we have worked to convert our second-generation SHERLOCK assays to the colorimetric lateral flow format. The recurrence of background in these assays sparked conversations with Scientists at Sherlock Biosciences who are actively developing cartridge-based, at-home SHERLOCK diagnostic assays for human infectious disease. These discussions led to the submission of a Phase I USDA SBIR grant in which we will work with Sherlock Biosciences to convert our WSSV assay to their field-deployable format. This grant was selected for funding with a start date of July 1, 2023.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Sullivan, T. J., Dhar, A. K., Cruz-Flores, R. and Bodnar, A. G. (2019) Rapid, CRISPR-based, field-deployable detection of White Spot Syndrome Virus in shrimp. Scientific Reports 9(1), 19702. https://doi.org/10.1038/s41598-019-56170-y
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Major, S.R., Harke, M.J., Cruz-Flores, R., Dhar, A.K., Bodnar, A.G. and Wanamaker S.A. (2023) Rapid detection of DNA and RNA shrimp viruses using CRISPR-based diagnostics. Applied and Environmental Microbiology. 0:e02151-02122. https://journals.asm.org/doi/10.1128/aem.02151-22
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Timothy Sullivan, Arun Dhar, Roberto Cruz-Flores, James Collins, Andrea Bodnar
CRISPR-Based diagnostic for rapid detection of White Spot Syndrome Virus in shrimp. 2020 Aquaculture America Conference, Honolulu, HI
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Shelly A. Wanamaker, Samuel R. Major, Arun Dhar, Andrea G. Bodnar
CRISPR-based diagnostics for disease detection in shrimp. 2022 Aquaculture Triennial Conference, San Diego, CA
|
Progress 05/07/21 to 05/06/22
Outputs
Target Audience:Co-PDDr. Arun Dhar and Research Scientist Dr. Shelly Trigg participated in the Aquaculture 2022 triennial conference, February 28 - March 4, in San Diego, CA where they presented the results of this project to the aquaculture community. Dr. Trigg presented a talk "CRISPR-based diagnostics for disease detection in shrimp" as part of the Shrimp Health and Disease session co-chaired by Dr. Dhar. Changes/Problems:The impacts of the COVID-19 pandemic and departure of key personnel resulted in significant delays in experimental progress and the submission of a "Change of Objectives" request which was approved in February 2022. Experimental progress was further delayed by unforeseen technical challenges associated with a key enzyme needed for the CRISPR SHERLOCK assays. The solution required a complete redesign of the diagnostic assays for both WSSV and TSV, to use loop-mediated amplification (LAMP) and a Cas12b detection system. The Cas12b enzyme is not commercially available and therefore was custom ordered from GenScript®, arriving during the first month of our first no-cost extension period (the current project period). During the current project period we did not encounter any significant challenges and have successfully designed, optimized, and validated thesecond-generation SHERLOCK assays for both WSSV and TSV completing the first two objectives of this project. We requested, and have been granted, a second no-cost extension to complete the remaining objective of this project and to prepare the results of the WSSV and TSV SHERLOCK assays for peer-review publication What opportunities for training and professional development has the project provided?Under the mentorship of the PD, Dr. Andrea Bodnar, Research Scientist Dr. Shelly Trigg has led the project to completion of Objectives 1 and 2, working with Research Associate Samuel Major. Dr. Trigg presented the work from this project at the international triennial Aquaculture Conference in San Diego February 28 - March 4 in a session chaired by the co-PD Dr. Arun Dhar. Dr. Trigg has also gained teaching experience through presenting work from this project as a guest lecturer for an undergraduate evolutionary and ecological genomics course at Northeastern University in Boston and has presented this work as part of the seminar series at the Bigelow Laboratory for Ocean Sciences, a non-profit research institute in Maine. PD, Dr. Bodnar has presented the results of this project as part of a guest lecture for NOAA's Northwest Fisheries Science Center and for the Darwin Festival at Salem State University. Dr. Trigg has gained experience forming and managing partnerships with industry including New England Biolabs, a biotech company who are experts in LAMP and Cas reagents, and Sherlock Biosciences, a start-up company founded in 2019 to use the SHERLOCK platform to revolutionize molecular diagnostics. Dr. Trigg and Samuel Major have also been participating as mentors at the Gloucester Biotechnology Academy where they have been able to share the work from this project with students preparing for careers in the biotechnology field. How have the results been disseminated to communities of interest?The results have been disseminated through presentationsat the Aquaculture 2022 conference as well asacademic and research organizations outlined below. Through these activities we have been able to reach a broad audience including aquaculture professionals and researchers (Aquaculture 2022, NOAA, Bigelow Labs), undergraduate and graduate students (Northeastern University and Salem State University), and the general public (Darwin Festival). Trigg SA, Major SR, Cruz-Flores R, Dhar A, and Bodnar A. CRISPR-based diagnostics for disease detection in shrimp. Aquaculture 2022 triennial conference. February 28 - March 4, 2022, Town and Country Conference Center San Diego, CA. Trigg, SA. Genomic tools for investigating oceans and human health. Guest lecture for Foundations in Ecological and Evolutionary Genomics (EEMB 1106) Spring 2022. March 30, 2022, Northeastern University, Boston, MA. Trigg, SA. Harnessing the power of genomics technologies for investigating marine animal health. Invited seminar for Northeastern University Marine Science Center Seminar Series. October 7, 2021 Trigg, SA. Genomic tools for investigating marine animal health. Bigelow Laboratory for Ocean Sciences Spring Seminar Series. April 7, 2022, Bigelow Laboratory for Ocean Sciences, East Boothbay, ME. Bodnar, AG. Enabling a New Era of Ocean Exploration and Discovery with Marine Genomics. Invited lecture for the Darwin Festival at Salem State University Feb 9, 2022 Bodnar, AG. Enabling a New Era of Ocean Discovery and Understanding at the Gloucester Marine Genomics Institute. Invited lecture for NOAA Northwest Fisheries Science Center, Feb 11, 2022 What do you plan to do during the next reporting period to accomplish the goals?The next project period will be focused on completing the third objective, to adapt the SHERLOCK assays to lateral flow strips as an initial step to developing a field-deployable format. This will involve converting the reporting system from fluorescence to colorimetric reporting using a custom reporter compatible with the lateral flow "hybridetect" test strips from Milenia Biotec (https://www.milenia-biotec.de/en/). This approach uses the FITC-antibody-gold nanoparticle chemistry to bind biotin in both cleaved and non-cleaved reporters from the SHERLOCK reaction producing horizontal lines to indicate the presence of reporter (control band) and the presence of the target (cleaved reporter; test band). Validation of the lateral flow reactions will be tested with shrimp infected with target virus, pathogen free shrimp, and shrimp infected with non-target pathogens to ensure accuracy and sensitivity. To eliminate the need for complex equipment in the sample processing step we will test a variety of simple nucleic acid extraction procedures like the paper matrix approach used with our first generation WSSV SHERLOCK assay (Sullivan at al. 2019 Scientific Reports9(1), 19702. https://doi.org/10.1038/s41598-019-56170-y). Finally, we will prepare the results of the WSSV and TSV SHERLOCK assays for peer-review publication and prepare the final USDA project report.
Impacts
What was accomplished under these goals?
Objective 1 - Design CRISPR assays to detect WSSV and TSV: Fifteen different sets of LAMP primers were designed to target four different gene regions in the WSSV genome and two different gene regions in the TSV genome. LAMP primer sets were screened for speed and sensitivity using synthetic viral target, and top performing sets were selected for testing with Cas12b. For WSSV and TSV, more than 20 different guide RNAs were tested in combination with selected LAMP primer sets for specificity, and reaction components were systematically optimized for highest sensitivity and reproducibility using fluorescence readout. The best performing primer and guide RNA combinations were used to generate standard curves using known amounts of synthetic viral target and demonstrated a reliable detection level of 10 viral copies for TSV and 100 viral copies for WSSV. These levels are comparable to, or better than, those achieved with standard OIE approved molecular techniques (qPCR and RT-qPCR). Objective 2 - Validate these assays against the standard molecular techniques (qPCR and RT-qPCR) for veterinary diagnostics: In previous project periods, co-PDArun Dhar conducted viral challenge experiments on Litopenaeus vannamei with WSSV and TSV. Infection status was measured with H&E histology and viral quantification using the OIE accepted methodologies (qPCR and RT-qPCR). The DNA and RNA samples from infected shrimp used for qPCR (WSSV) and RT-qPCR (TSV) were shipped to GMGI to use in the validation experiments for the SHERLOCK assays. In this project period, validation of the LAMP/Cas12b SHERLOCK assays for WSSV and TSV was completed with 35 samples validated for WSSV and 35 samples validated for TSV, and all positives were consistent with qPCR results. There was not a strong correlation of viral quantities estimated by SHERLOCK with those estimated by qPCR; however, this is not surprising given that all assays target different genomic regions which could contribute to different amplification efficiencies, differences in standard curve input (qPCR used a plasmid and SHERLOCK used a blunt amplicon), differences in the effect of nuclei acid input amount on the assays (amount of nucleic acid input was normalized for the SHERLOCK assays, input for qPCR assays was not normalized). To ensure no cross-reactivity for other common pathogens, we tested specificity of the SHERLOCK assays using DNA and RNA extracted from shrimp infected with Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV), Infectious Myonecrosis Virus (IMNV), Enterocytozoon hepatopenaei (EHP), and Vibrio parahaemolyticus which causes Acute Hepatopancreatic Necrosis Diseases (AHPND)]. Additionally, WSSV infected shrimp were tested in the TSV SHERLOCK assay and TSV infected shrimp were tested in the WSSV SHERLOCK assay. Of the 10 samples (2 individuals per alternative virus) used in specificity testing, no sample tested positive in the WSSV or TSV SHERLOCK assays. To estimate the false positive rate, DNA and RNA was extracted from 10 individual pathogen-free shrimp and tested in WSSV and TSV SHERLOCK assays which resulted in no positive detections. Objective 3 - The impacts of the COVID-19 pandemic and departure of key personnel resulted in significant delays in experimental progress and the submission of a "Change of Objectives" request which was approved in February 2022 in which the third objective was changed to:Adapt the SHERLOCK assays to lateral flow strips as an initial step to developing a field-deployable format. Our effort in the current project period has been on ensuring that the second-generation, LAMP/Cas12b SHERLOCK assays were robust and reproducible prior to converting to a colorimetric lateral flow format. Now that we have achieved high quality assays, the focus for the next project period under our second no-cost extension will be to complete Objective 3.
Publications
|
Progress 05/07/20 to 05/06/21
Outputs
Target Audience:
Nothing Reported
Changes/Problems:There were several major problems that delayed progress during this project performance period, including the COVID19 pandemic, departure of key personnel, and technical challenges associated with one of the key components of our original assay. The COVID-19 pandemic led to State mandated shut-down of non-essential businesses and institutional restrictions that prevented or limited access to the lab from March until August 2020, resulting in a significant disruption in experimental work. The departure of the project PD, Tim Sullivan (in June 2020), and departure of the Research Associate dedicated to this project, Veronica Pereira (in August 2020), both of whom joined the USDA. In order to keep the project moving forward, Research Associate Samuel Major, shifted 50% of his effort from his assigned project to this USDA-funded project. With the easing of COVID-related restrictions and increased access to the lab, Samuel was able to make good progress on reproducing the WSSV assay developed in the first year of the project and establishing a two-step TSV assay. However, Samuel encountered an unexpected technical problem that further delayed progress. The technical problem was related to variation in the recombinase polymerase amplification (RPA), one of the key steps of the original CRISPR-based SHERLOCK assay. We discovered that some batches of the recombinase polymerase enzyme mix, currently only available from TwistDx™, exhibited RNase activity which was particularly problematic for the Cas13a RNA-based detection system, resulting in high background in the SHERLOCK assay. This discovery led to the redesigning of both the WSSV and TSV assays to use loop-mediated amplification (LAMP) instead of RPA, and Cas12b for DNA-based detection. This redesign strategy was inspired by the successful development of the STOPCovid SHERLOCK diagnostic test for SARS-CoV-2 as well as other recent SHERLOCK applications. As demonstrated in the work of others, we feel confident that switching to a LAMP-Cas12b approach will solve the problem that we encountered and also improve the sensitivity of the assay. In March 2021, GMGI recruited a new Research Scientist, Dr. Shelly Trigg, who is focused on completing the project objectives in the next project period under the requested no-cost extension. What opportunities for training and professional development has the project provided?The original PD on this project, Tim Sullivan, left GMGI in June to join the USDA as a National Program Leader of Animal Production Systems. This provided an opportunity for Andrea Bodnar (GMGI's Science Director) to assume leadership of this project and provide mentorship to two Research Associates: Veronica Pereira and Samuel Major. Veronica, participated in this project period from May until August when she left GMGI to join the USDA in Harrisburg, PA as a Survey Statistician. Samuel began working on this project in July. Both Veronica and Samuel were trained in the theoretical and technical aspects of designing and executing the CRISPR-based diagnostic tests with a focus on the TSV assay. This included designing primers for RPA, and crRNA for CRISPR-based molecular detection, executing both fluorescence and lateral-flow colorimetric assays, and troubleshooting problems related to variability in sensitivity. This project also provided training for a post-doctoral scientist under the mentorship of co-PD Arun Dhar at the University of Arizona. In this project period, post-doctoral scientist, Roberto Cruz-Flores, conducted the TSV challenge experiments and evaluation of viral infection using histology and RT-qPCR. Dr. Cruz-Flores left the project in February to accept a Principal Scientist position at the Center for Scientific Research and Higher Education (CICESE) in Ensenada, Mexico. In March 2021, GMGI recruited a new Research Scientist, Dr. Shelly Trigg, who is now fully trained in the SHERLOCK technology and will focus on completing the project objectives over the next year under the mentorship of the PD, Andrea Bodnar. 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?Given the delays in progress due to the COVID19 pandemic, departure of key personnel, and the technical challenges associated with the RPA reagents, we have requested a 12 month no-cost extension to complete the objectives of this project. In this time, we will complete the optimization of our next generation SHERLOCK assays for TSV and WSSV using LAMP amplification and the crRNA/Cas12b DNA detection system. Following optimization, we will test the sensitivity, accuracy, and reproducibility using both fluorescence and lateral flow readouts. The final SHERLOCK assays will then be used to test the RNA from the viral challenge experiments conducted in the previous project periods to correlate the SHERLOCK results with that of OIE accepted protocols for viral detection (qPCR and RT-qPCR). Following these validation steps, we will convert the final SHERLOCK assays for WSSV and TSV to the field test kit format based on a lyophilized lateral flow test strip. The kits will be first evaluated in blind ring tests with samples supplied by co-PD Arun Dhar and, upon successful completion of the blind ring tests, the test kits will be sent to the aquaculture partners to obtain feedback on effectiveness and ease of use in the field. During the final year of the project, we will prepare the results of the next generation WSSV and TSV SHERLOCK assays for peer-review publication. We anticipate presenting the latest results of this project at the Triennial International Aquaculture 2022 Conference in San Diego, CA in February which includes participants from the World Aquaculture Society, American Fisheries Society, National Shellfisheries Association, U.S. Aquaculture Society, National Aquaculture Association and Aquaculture Suppliers Association.
Impacts
What was accomplished under these goals?
Impact: The occurrence of highly virulent diseases is one of the most significant impediments to the profitability and success of aquaculture production. For shrimp farming alone, pathogens such as White Spot Syndrome Virus (WSSV) and Taura Syndrome Virus (TSV) have cost global producers billions of dollars since their detection in the 1990s. The largescale impact of disease on production makes rapid and accurate diagnosis of infection critically important for disease management. For this reason, we have begun to develop new molecular diagnostic tools for rapid, sensitive, cost-effective tests that can be conducted in the field without sophisticated equipment or specialized training. This approach will empower farmers with the ability to rapidly respond to disease outbreaks, mitigating spread and minimizing losses without dependence on dedicated laboratory facilities. During this project, we have designed two cutting-edge diagnostic assays that use a CRISPR-based molecular approach to enable detection of WSSV and TSV. The test designed for WSSV (a virulent DNA virus) can detect as low as a single DNA viral copy in as little as 1 hour, which is both more rapid and more sensitive than all other available methods for detection of this virus. We demonstrated that this WSSV assay was highly specific and did not detect other common shrimp pathogens including TSV, IHHNV, IMNV, EHP, and AHPND. Further, the results of the CRISPR-based test were highly correlated with results from the currently accepted methodology for viral detection (qPCR). The test designed for TSV (an RNA virus) is currently conducted as a 2-step protocol and is sensitive down to 100 synthetic viral RNA copies which is comparable to current methodologies. Both the WSSV and TSV CRISPR-based diagnostic tests have been designed with fluorescence readout for quantitative results conducted in a laboratory, and using a paper strip colorimetric approach to report a positive or negative test result in the field. We are currently working to maximize the robustness and reproducibility of these assays to ensure their performance in the aquaculture field setting. Once complete, these novel molecular tests will enable a cost-effective approach to increase disease surveillance by aquaculture producers and provide accurate and sensitive diagnostic results for proactive disease management resulting in increased sustainability and profitability of production across the US and globally. Objective 1 - Design CRISPR assays to detect WSSV and TSV: In the first project period, we designed, validated and published a CRISPR-based SHERLOCK diagnostic test for WSSV. In the current project period, the focus has been on designing and validating a SHERLOCK diagnostic assay for TSV. TSV is an RNA virus and therefore requires the addition of a reverse transcription (RT) step prior to the amplification and CRISPR detection as part of the SHERLOCK assay. In this project period the recombinase polymerase amplification (RPA) primers and CRISPR probes designed in the first year of the project were tested on a synthetic TSV RNA target to identify the combination that produced the most sensitive reaction. Using a two-step protocol in which the RT-RPA reaction was conducted prior to the CRISPR/Cas13a detection step, our best primers and probe produced a standard curve with detection down to 100 synthetic viral RNA copies in both fluorescence and lateral flow formats. However, reproducing this level of sensitivity was hindered by certain batches of the RPA polymerase that contained contaminating RNases, which resulted in reduced target detection as well as high background cleavage of the RNA reporter used by the Cas13a detection system. Throughout the development of the TSV assay, we have been in communication with scientists at Sherlock Biosciences who recently developed a SHERLOCK assay for SARS-CoV-2, another RNA virus. Their solution to create a reliable one-step reaction was to use loop-mediated isothermal amplification (LAMP) instead of RPA and replace Cas13a with Cas12b which uses a DNA-based detection system. With this knowledge in hand, we designed and tested four sets of LAMP primers for TSV, three of which showed positive results in an RT-LAMP assay. Our custom order for Cas12b, which is not yet commercially available, will be arrivingfrom GenScript®bymid-May, and will be used to test the crRNA targets designed to detect the amplified products. This next generation of our SHERLOCK assay for TSV detection that should overcome the technical hurdles we have encountered. Objective 2 - Validate these assays against the standard molecular techniques for veterinary diagnostics (qPCR and RT-qPCR): In preparation for the validation of the new TSV SHERLOCK assay, two viral challenge experiments were conducted. In the first bioassay, P. vannamei (Kona line) were injected with TSV inoculum intramuscularly and five moribund animals were collected for H&E histology and TSV quantification by real-time quantitative PCR (RT-qPCR). In the second bioassay, a commercial line of P. vannamei that is known to be resistant to TSV was injected intramuscularly with TSV inoculum, and thirty animals were sampled following experimental challenge for histopathology and RT-qPCR assays. Upon TSV experimental challenge, P. vannamei (Kona line) showed mortalities and clinical signs of Taura syndrome. However, TSV resistant shrimp did not show mortality or any signs of disease. Histopathological evaluation of the challenged shrimp showed pathognomonic lesions characteristic of TSV infection in samples derived from the Kona line, while no lesions were detected in the TSV resistant shrimp. TSV was detected by RT-qPCR following the OIE recommended protocol in 31 out of the 35 challenged shrimp, indicating a high level of sensitivity of RT-qPCR over histopathological methods. RNA samples used for RT-qPCR were shipped to GMGI where they are being stored at -80°C and will be tested using the final version of the TSV SHERLOCK assay for direct comparison of TSV quantification using the SHERLOCK method and the OIE-recommended RT-qPCR method. Objective 3 - Employ on-site testing by aquaculture farmers: In order to be effectively executed in the aquaculture field setting, SHERLOCK diagnostic tests must be highly robust and reproducible. Therefore, prior to creating a field-based kit, we tested the reproducibility of the WSSV SHERLOCK assay that was developed in the first year of this project. Experiments in this project period were unable to reproduce the high level of sensitivity originally reported for the WSSV SHERLOCK assay, which was due to RNase activity associated with certain batches of the RPA enzyme mix causing degradation of the target RNA and non-specific cleavage of the RNA reporter. In order to address these challenges, we have designed amplification primers for LAMP instead of RPA and have explored using an alternative Cas detection system (Cas12b) which uses a DNA-based reporting system. LAMP primers were designed to four different target regions of the WSSV genome and threesuccessfully generated amplified products. Our custom order for Cas12b, which is not yet commercially available, will be arrivingfrom GenScript®by mid-May, and will be used to test the crRNA targets designed to detect the amplified products.
Publications
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Progress 05/07/19 to 05/06/20
Outputs
Target Audience:We have engaged multiple audiences during the current period. We have done this in threeways. First, we have published the results of the development of our SHERLOCK diagnostic assay for the rapid detection of White Spot Syndrome Virus infecting Pacific white shrimp in the journal Scientific Reports. We chose this journal because it is well respected in academic circles, but also because it is an open access journal. This allows non-academic audiences the ability to freely access the information without any sort of paywall. Second, the publication of this data was highlighted in the March 2020 newsletter that GMGI sends to its network of donors, friends, and supporters which include hundreds of individuals aroung the greater new england area as well as others throughout the US. Third, Dr. Sullivan presented the development of this diagnostic test during an oral presentation entitled "CRISPR-BASED DIAGNOSTICS FOR RAPID DETECTION OF WHITE SPOT SYNDROME VIRUS IN SHRIMP" at the 2020 Aquaculture America meeting held in Honolulu HI from February 9th 2020 to February 12th 2020. This meeting includes a broad mix of academic researchers, NGO members, private for-profit companies, and aquatic animal producers all focused on aspects of aquaculture. This presentation took place in a session on Shrimp Health chaired by Dr. Dhar (coPI on this project) and was attended by a diverse group of US and international attendees. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?GMGI hired a new research associate (Veronica Pereira) five months ago. In the past five months Veronica has been fully trained in the design and development of SHERLOCK assays including both the RPA amplification primers and the crRNAs that program the activation of CRISPR enzymes. She is now taking the lead on experiments to develop and validate the TSV diagnostic test. In the area of professional development, Drs. Sullivan and Dhar both attended the Aquaculture America conference held February 9th to 12th in Honolulu HI. During the meeting Dr. Sullivan also attended a workshop on Fish Health Management. How have the results been disseminated to communities of interest?We have disseminated our results in three ways. First, we published our WSSV diagnostic assay development and validation in the journal Scientific Reports. We chose ths journal because it is fully open access allowing for any person to access the results without cost to them. Second, we distributed a summary of our work in the March issue of GMGI's newletter that has a broad reach wihtin New England and around the country. Lastly, Dr. Sullivan presented the design, development, and application of our WSSV SHERLOCK diagnostic test at the 2020 Aquaculture America meeting in Honolulu HI in a session on Shrimp Health to a room of US and International researchers, aquaculture enterprises, and producers. What do you plan to do during the next reporting period to accomplish the goals?In the area of outreach, Dr. Sullivan will be giving a public lecture in late April to GMGI donors, friends, and the public on the application of CRISPR technologies to diagnostics, animal health, and aquatic animal production along side a representative of Arbor Biotechnologies (which develops new crispr enzymes) and Dr. Andrea Bodnar who uses CRISPR tools to transform animal models. Dr. Sullivan and Veronica Pereira will also be making a informational poster for public consumtion that outlines the work done and outcomes of the current project. Dr. Sullivan is also planning to present the combined WSSV and TSV results at the upcoming 2020 American Fishery Society meeting in Colombus Ohio. In the area of research, we will be engaged in fourprimary acitivies. First, Veronica Pereira will be continuing to validate our TSV diagnostic assay in the laboratory as well as converting it from flourescent to colorimetric composition. Second, Veronica Pereira and Dr. Sullivan will be testing our set of challenge time course sample with the SHERLOCK WSSV assay in a quantitative fashion. These sampels were generated at the Aquaculture Pathology Laboratory by sampling infection challenged shrimp at 3, 6, 9, 12, 24, 48, 72, and 96 hours post-infection. The quantification results and diagnostic infection results will be compared to histology and qPCR to determine which approach is most sensitive and can diagnose earliest on a large-scale. The challenge has already been completed. Third, Dr. Sullivan will be traveling to the Aquaculture Pathology Laboratory to test live shirmp samples with our field-based approach. This will also allow for training the Aquaculture Pathology Laboratory staff on this new approach. Lastly, we are in the process of designing what our test kit for volunteers will look like and by the summer we hope to be assembling these kits for distribution to our original test group as well as new volunteers recruited during the Aquaculture America meeting.
Impacts
What was accomplished under these goals?
Impact: The occurence of highly virulent diseases is one of the most impactful impediments tothe profitability and success of aquaculture production, especially for species that lack adaptive immune systems (such as shrimp). For instance, in white shrimp production, WSSV and TSV have cost global producers billions of dollars since their detection in the 1990s. The largescale impact of disease on production makes rapid and accurate diagnosis of infection critically important fordisease management. For this reason, we have begun to develop new diagnostic tools that could allow for field-deployable testing by lay people as opposed to only laboratory testing by advanced professionals. During the first project period, we have been able to design two cutting-edge, revolutionary diagnostic assays that use new CRISPR-based molecular approaches to enable detection of viruses by DNA sequence without needing electricity, large dedicated laboratory facilitites, sophisticated equipment, or specialized training. The approach that we have validated for WSSV (a virulent DNA virus of shirmp) can detect as low as a single DNA viral copy in as little as 1 hour, which is both more rapid and more sensitive than all other available methods for detection of these viruses. For our WSSV assay, we have been able to show that beyond single copy detection, our assay does not detect any other common shirmp pathogensincluding TSV, IHHNV, IMNV, EHP, and AHPND and that when we quantify the amount of virus with our approach the results are highly correlated (r=0.93, p=1.4e-15)and similar to those generated by qPCR (the currently accepted methodology). After validating ourtest, we converted it from a flourescent method to a strip-based colorimetric approach that uses a paper strip to report a positive or negative test result. We then optomized a field extraction method that extracts DNA/RNA in a few minutes using filter paper strips. In total, this allowed for a diagnostic process that can proceed from shrimp tissue to a positive or negative diagnosis in ~60 minutes with no electricity. This opens the door for truly field-deployable diagnosis of aquaculture infections and hopefully will spur a increase in disease surveillance by aquaculture laboratories and producers as well as an increase in proactive disease management and increases in sustainability and profitability of production across the US. Our objectives are: 1) To design CRISPR assays for the detection of WSSV and TSV strains in laboratory settings We are able to download whole genomes of WSSV and TSV and use these to design multiple sets of RPA primers as well as multiple sets of CRISPR guide RNAs (crRNAs) for the programming and activation of the CRISPR enzymes. We systematically tested each set of primers with each crRNA to arrive at a single set of each for WSSV and for TSV that showed maximum efficiency and activation, indicating a likely strong candidate for the diagnostic assay. In that these designs resulted in components that are successful at detecting these diseases, we consider this objective complete. 2) To validate these assays against the standard molecular techniques (qPCR and rt-qPCR) for veterinary diagnostics Dr. Sullivan has fully validated the WSSV assay. For the validation he engaged in 4 experiments. The first was togenerate a 5-point standard curve from 10 copies up to 100,000 viral copies. This curve was highly successful with strong linearity and a line of best fit ofy = 830117 × −268875,R2 = 0.988. Next, we took 20 nanograms of DNA from a single infected shirmp isolate and diltued it through a 10-fold serial dilution down to 20 attograms. These DNA amount were used as input into sherlock tests to determine the limit of sensitivity of our test. Our assay (based on an ANOVA and post-hoc tests) was able to detect down to 20 femtograms, which based on the estimated copy number of the individual was single copy detection. Our third test was to compare SHERLOCK to qPCR quantification. We found thatquantification of copy number for WSSV infected white shrimp with both qPCR and SHERLOCK assays was strongly correlated:r = 0.93 which was significant (p = 1.4e−15). Lastly, we tested our assay against other common pathogens, including:TSV,Vibriospp. causing AHPND, EHP, IHHNV, IMNV, and SPF (verified pathogen free) shrimp. Our assay did not show any detection signal for any of these other pathogens when compared to no template controls. Once our validation was complete we coverted our test from flourescent to colorimetric by changing the reporting molecule. Then we combined this test with a rapid field extraction method using filter paper. We then ran field-based or colorimetric tests with no template reactions, SPF shrimp samples and WSSV infected samples and our assays produced positive results only for WSSV infected samples. Following this we tested varying amounts of viral copy input, down to 10 copies and showed that our field-based test could detect down to between 10 and 100 copies. In total, our field-based assay was able to extract DNA, run a reaction, and generate a strip-based color read out in 60 minutes at room temperature with no electricity required. We are presently beginning to engage in the same sets of experiments with our TSV assay. 3) To employ on-site testing by aquaculture farmers We have begin designing what our test kit may look like and what the components involved will be as well as how they will be comprised and how the instructions for use will be composed. We have not produced the kit yet nor have we sent them as of yet.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Rapid, CRISPR-Based, Field-Deployable Detection Of White Spot Syndrome Virus In Shrimp
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
CRISPR-BASED DIAGNOSTICS FOR RAPID DETECTION OF WHITE SPOT SYNDROME VIRUS IN SHRIMP presented at 2020 Aquaculture America meeting in Honolulu HI
|
Progress 05/01/19 to 04/30/20
Outputs
Target Audience:We have engaged multiple audiences during the current period. We have done this in threeways. First, we have published the results of the development of our SHERLOCK diagnostic assay for the rapid detection of White Spot Syndrome Virus infecting Pacific white shrimp in the journal Scientific Reports. We chose this journal because it is well respected in academic circles, but also because it is an open access journal. This allows non-academic audiences the ability to freely access the information without any sort of paywall. Second, the publication of this data was highlighted in the March 2020 newsletter that GMGI sends to its network of donors, friends, and supporters which include hundreds of individuals aroung the greater new england area as well as others throughout the US. Third, Dr. Sullivan presented the development of this diagnostic test during an oral presentation entitled "CRISPR-BASED DIAGNOSTICS FOR RAPID DETECTION OF WHITE SPOT SYNDROME VIRUS IN SHRIMP" at the 2020 Aquaculture America meeting held in Honolulu HI from February 9th 2020 to February 12th 2020. This meeting includes a broad mix of academic researchers, NGO members, private for-profit companies, and aquatic animal producers all focused on aspects of aquaculture. This presentation took place in a session on Shrimp Health chaired by Dr. Dhar (coPI on this project) and was attended by a diverse group of US and international attendees. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?GMGI hired a new research associate (Veronica Pereira) five months ago. In the past five months Veronica has been fully trained in the design and development of SHERLOCK assays including both the RPA amplification primers and the crRNAs that program the activation of CRISPR enzymes. She is now taking the lead on experiments to develop and validate the TSV diagnostic test. In the area of professional development, Drs. Sullivan and Dhar both attended the Aquaculture America conference held February 9th to 12th in Honolulu HI. During the meeting Dr. Sullivan also attended a workshop on Fish Health Management. How have the results been disseminated to communities of interest?We have disseminated our results in three ways. First, we published our WSSV diagnostic assay development and validation in the journal Scientific Reports. We chose ths journal because it is fully open access allowing for any person to access the results without cost to them. Second, we distributed a summary of our work in the March issue of GMGI's newletter that has a broad reach wihtin New England and around the country. Lastly, Dr. Sullivan presented the design, development, and application of our WSSV SHERLOCK diagnostic test at the 2020 Aquaculture America meeting in Honolulu HI in a session on Shrimp Health to a room of US and International researchers, aquaculture enterprises, and producers. What do you plan to do during the next reporting period to accomplish the goals?In the area of outreach, Dr. Sullivan will be giving a public lecture in late April to GMGI donors, friends, and the public on the application of CRISPR technologies to diagnostics, animal health, and aquatic animal production along side a representative of Arbor Biotechnologies (which develops new crispr enzymes) and Dr. Andrea Bodnar who uses CRISPR tools to transform animal models. Dr. Sullivan and Veronica Pereira will also be making a informational poster for public consumtion that outlines the work done and outcomes of the current project. Dr. Sullivan is also planning to present the combined WSSV and TSV results at the upcoming 2020 American Fishery Society meeting in Colombus Ohio. In the area of research, we will be engaged in fourprimary acitivies. First, Veronica Pereira will be continuing to validate our TSV diagnostic assay in the laboratory as well as converting it from flourescent to colorimetric composition. Second, Veronica Pereira and Dr. Sullivan will be testing our set of challenge time course sample with the SHERLOCK WSSV assay in a quantitative fashion. These sampels were generated at the Aquaculture Pathology Laboratory by sampling infection challenged shrimp at 3, 6, 9, 12, 24, 48, 72, and 96 hours post-infection. The quantification results and diagnostic infection results will be compared to histology and qPCR to determine which approach is most sensitive and can diagnose earliest on a large-scale. The challenge has already been completed. Third, Dr. Sullivan will be traveling to the Aquaculture Pathology Laboratory to test live shirmp samples with our field-based approach. This will also allow for training the Aquaculture Pathology Laboratory staff on this new approach. Lastly, we are in the process of designing what our test kit for volunteers will look like and by the summer we hope to be assembling these kits for distribution to our original test group as well as new volunteers recruited during the Aquaculture America meeting.
Impacts
What was accomplished under these goals?
Impact: The occurence of highly virulent diseases is one of the most impactful impediments tothe profitability and success of aquaculture production, especially for species that lack adaptive immune systems (such as shrimp). For instance, in white shrimp production, WSSV and TSV have cost global producers billions of dollars since their detection in the 1990s. The largescale impact of disease on production makes rapid and accurate diagnosis of infection critically important fordisease management. For this reason, we have begun to develop new diagnostic tools that could allow for field-deployable testing by lay people as opposed to only laboratory testing by advanced professionals. During the first project period, we have been able to design two cutting-edge, revolutionary diagnostic assays that use new CRISPR-based molecular approaches to enable detection of viruses by DNA sequence without needing electricity, large dedicated laboratory facilitites, sophisticated equipment, or specialized training. The approach that we have validated for WSSV (a virulent DNA virus of shirmp) can detect as low as a single DNA viral copy in as little as 1 hour, which is both more rapid and more sensitive than all other available methods for detection of these viruses. For our WSSV assay, we have been able to show that beyond single copy detection, our assay does not detect any other common shirmp pathogensincluding TSV, IHHNV, IMNV, EHP, and AHPND and that when we quantify the amount of virus with our approach the results are highly correlated (r=0.93, p=1.4e-15)and similar to those generated by qPCR (the currently accepted methodology). After validating ourtest, we converted it from a flourescent method to a strip-based colorimetric approach that uses a paper strip to report a positive or negative test result. We then optomized a field extraction method that extracts DNA/RNA in a few minutes using filter paper strips. In total, this allowed for a diagnostic process that can proceed from shrimp tissue to a positive or negative diagnosis in ~60 minutes with no electricity. This opens the door for truly field-deployable diagnosis of aquaculture infections and hopefully will spur a increase in disease surveillance by aquaculture laboratories and producers as well as an increase in proactive disease management and increases in sustainability and profitability of production across the US. Our objectives are: 1) To design CRISPR assays for the detection of WSSV and TSV strains in laboratory settings We are able to download whole genomes of WSSV and TSV and use these to design multiple sets of RPA primers as well as multiple sets of CRISPR guide RNAs (crRNAs) for the programming and activation of the CRISPR enzymes. We systematically tested each set of primers with each crRNA to arrive at a single set of each for WSSV and for TSV that showed maximum efficiency and activation, indicating a likely strong candidate for the diagnostic assay. In that these designs resulted in components that are successful at detecting these diseases, we consider this objective complete. 2) To validate these assays against the standard molecular techniques (qPCR and rt-qPCR) for veterinary diagnostics Dr. Sullivan has fully validated the WSSV assay. For the validation he engaged in 4 experiments. The first was togenerate a 5-point standard curve from 10 copies up to 100,000 viral copies. This curve was highly successful with strong linearity and a line of best fit ofy = 830117 × −268875,R2 = 0.988. Next, we took 20 nanograms of DNA from a single infected shirmp isolate and diltued it through a 10-fold serial dilution down to 20 attograms. These DNA amount were used as input into sherlock tests to determine the limit of sensitivity of our test. Our assay (based on an ANOVA and post-hoc tests) was able to detect down to 20 femtograms, which based on the estimated copy number of the individual was single copy detection. Our third test was to compare SHERLOCK to qPCR quantification. We found thatquantification of copy number for WSSV infected white shrimp with both qPCR and SHERLOCK assays was strongly correlated:r = 0.93 which was significant (p = 1.4e−15). Lastly, we tested our assay against other common pathogens, including:TSV,Vibriospp. causing AHPND, EHP, IHHNV, IMNV, and SPF (verified pathogen free) shrimp. Our assay did not show any detection signal for any of these other pathogens when compared to no template controls. Once our validation was complete we coverted our test from flourescent to colorimetric by changing the reporting molecule. Then we combined this test with a rapid field extraction method using filter paper. We then ran field-based or colorimetric tests with no template reactions, SPF shrimp samples and WSSV infected samples and our assays produced positive results only for WSSV infected samples. Following this we tested varying amounts of viral copy input, down to 10 copies and showed that our field-based test could detect down to between 10 and 100 copies. In total, our field-based assay was able to extract DNA, run a reaction, and generate a strip-based color read out in 60 minutes at room temperature with no electricity required. We are presently beginning to engage in the same sets of experiments with our TSV assay. 3) To employ on-site testing by aquaculture farmers We have begin designing what our test kit may look like and what the components involved will be as well as how they will be comprised and how the instructions for use will be composed. We have not produced the kit yet nor have we sent them as of yet.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Rapid, CRISPR-Based, Field-Deployable Detection Of White Spot Syndrome Virus In Shrimp
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
CRISPR-BASED DIAGNOSTICS FOR RAPID DETECTION OF WHITE SPOT SYNDROME VIRUS IN SHRIMP presented at 2020 Aquaculture America meeting in Honolulu HI
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