Performing Department
(N/A)
Non Technical Summary
Disease outbreaks are being increasingly recognized as a significant constraint to aquaculture production and trade and are affecting economic development. Monitoring fish health for early detection of diseases is paramount to secure a safe and sustainable aquaculture industry. Environmental DNA (eDNA) is a promising tool that has not yet delivered its disruptive potential for aquaculture health monitoring. While sensitive and reliable eDNA detection techniques such as PCR are widely available, the major limitation today is the quality and reliability of eDNA sampling and purification techniques in aquaculture environments. During Phase I, we demonstrated the technical feasibility of a novel nanosorbent that captures 20 times more eDNA thus eliminating false results, is 5 times faster and 80% less expensive. In this Phase II, we will (1) conduct additional research to understand the sample volume needed for a reliable detection of aquatic pathogens, (2) develop a housing/delivery system for the nanosorbent to build a working prototype that simplifies and streamlines the eDNA workflow, and (3) conduct an independent testing and validation of the prototype by academic and industry partners. The new technology is expected to significantly enhance fish health monitoring, thus enabling a better management of sustainable and productive fisheries and aquaculture, and improving food safety and security. Such development would also trigger a wide adoption of the eDNA technology by public and private stakeholders, and a rapid growth of the eDNA testing market. In addition, the proposed technology would empower citizen science programs for bioconservation and monitoring of aquatic invasive species.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
The goal of the SBIR Phase II program is to conduct additional research to enhance our understating of sample volume requirements for eDNA detection, then develop and demonstrate a working prototype or minimum viable product for the eDNA collection and purification technology, thus establishing its commercial viability. Building on the successful technical feasibility of the nanosorbents in Phase I, Phase II research and development effort will focus on the development of the complete prototype and workflow and their testing and validation by third-party partners and lead customers. The project specific objectives are:Specific Objective 1: Understanding and establishing the optimum sample volume for eDNA pathogen testing in aquacultureSpecific Objective 2: Developing housing/delivery system for the nanosorbentsSpecific Objective 3: Demonstrating of the minimum viable product in field conditionsSpecific Objective 4: Testing and validation of the MVP by third-party partners and lead customersSpecific Objective 5: Developing and testing a system for the scale-up of nanosorbent manufacturing
Project Methods
Frontline Biotechnologies Inc. is a science- and evidence-based company. The research and development approach is designed to answer the technical questions raised earlier and achieve the phase II objectives by relaying on rigorous scientific experiments and reproducible data and statistical analysis, and confirmed by third-party independent demonstration. The work plan follows 5 major tasks:Task 1- Studying and establishing the optimum sample volume for eDNA testing in aquaculture: A large number of studies on eDNA revealed the necessity of collecting large volumes of water (1-10 L) to increase the probability of species detection using eDNA.22-24 The widely adopted protocols for eDNA sample collection produced by the U.S. Geological Survey and the National Genomics Center for Wildlife and Fish Conservation (1-10 L sample volume) effectively collect up to 500 times more DNA than protocols that sample less than 100 mL of water.Methods and techniques: Wastewater samples with different volumes ranging from 10 mL to 10 L will be collected, and the samples processed for nucleic acid extraction using current methods and the Frontline's technology. Target viruses will be detected using RT-PCR technique to establish a correlation of the sample volume versus the number of viral copies detected per mL. This will help establish a protocol of an optimal use of Frontline's technology.Task 2 - Developing housing/delivery systems for biosampling and purification: A sample collection device will be designed to perform multiple functions in a single cartridge, named eBiosampler (Figure 4). The cartridge will be designed to filter up to 3 L of water in the field, and perform neutralization/lysis of the biological material immediately after collection, sample preservation inside the cartridge during transport, and extraction/purification of the target nucleic acids in the lab for RT-PCR analysis. The cartridge can connect from one side to a syringe or pumping system for water filtration, and connect to a spin column (Biospin) and collection/centrifuge tubes in the other side. This device will enable a single and streamlined eDNA workflow.Method and techniques: AutoCAD modeling and design software will be used to design the biosampling and purification cartridge, and Autodesk CFD will be used for computational fluid dynamic simulations to optimize flow inside the cartridge. The first prototype will be fabricated using 3D printing available at the University of Minnesota Medical Device Center. After initial testing and adjustment, the final design will be send to our prototyping partner Xometry Inc.kl to fabricate the mold manufacture some samples using industrial plastics.Task 3 - In-house performance comparison study with the MVP: The final MVP prototypes (collection and purification system) including the cartridge (eBiosampler) and the Biospin hosting the nanocomposite sorbents and the accompanying buffer kit, will be first tested internally by Frontline's R&D team.Method and techniques:Sampling and filtration: An aquaculture site will be selected with our partners. The approach is to collect simultaneously multiple samples from the same site using a conventional approach and using Frontline's eDNA kit (MVP). The Frontline's kit will be used following a defined workflow. Using qPCR, we will compare eDNA copy number per sample and probability of detection between the two approaches. In a set of small experimental aquaculture sites, we will stock a known number of fish and spike the water with a known concentration of a virus or a microorganism of interest (Table 2), then sample soon after to create a challenging low eDNA scenario. At least two viruses or one virus and one parasite from the list in Table 2 will be used for this internal demonstration and performance evaluation. The primers will be designed for ITS region for each species and qPCR will be performed.eDNA amplification and quantification: Quantitative PCR (qPCR) will be performed using StepOnePlus Real-Time PCR System to amplify and quantify DNA sequences related to target species. Species selection was based on two main parameters: the importance of the species for the aquaculture industry, and the availability of PCR assays specific to the target species. eDNA amplification and quantification will be performed with at least 3 qPCR replicates per sample. When coupled with qPCR technology, eDNA analysis has proven to be more specific and more sensitive than traditional PCR, with a high probability of detection at concentrations as low as 0.5 target copies/µl. When dealing with high filtered volume of water and thus high DNA content, specificity is very important to prevent false negative in the detection of invasive species. The qPCR assays for the selected species will be based on protocols reported in literature with some modifications, using best practices and controls necessary for highly sensitive eDNA work.Data collection and analysis: Field tests will be evaluated by comparisons of mean eDNA copynumber per sample and proportion of samples above qPCR limits of detection (i.e, proportion of positives), with the one-sided hypothesis that our new kit will have higher levels of each than traditional single site samples. We suggest modest sample sizes will be sufficient because we expect substantial increases in eDNA detection probability and quantity, and small improvements would be unlikely to justify a new sampling kit. Based on power analyses for comparison of proportions, with proposed sample sizes of 20 we expect approximately 80% power to detect significant differences (at α = 0.1) if traditional samples detected one positive (5%) while the new kit detected 7 (40%) or more, whereas if traditional methods had 5 (25%) positives then our device would need at least 13 (65%) positives. Detection studies use variable numbers of qPCR replicates per water sample andvarying criteria for the number of replicates that need to be positive to declare the sample positive. We will further compare our detection probabilities based on criteria of 1, 2, or all 3 replicates being positive.Task 4 - Third-party independent testing and validation of the MVP: Following a successful internal testing and validation of the MVP, 1000 prototypes will be manufactured and sent freely to our partners (200 kits per partner). These partners expressed interest to conduct independent comparison study to assess the performance of the MVP as compare it to their current practice. They will also provide feedback on potential improvement before commercial launch of the product. The industry partner has also expressed interest to purchase the product after a successful evaluation (see Interest and Collaboration letters).Task 5- Development and testing of a system for the scale-up of nanosorbent manufacturing: During Phase 1, a small lab-scale system was used for the preparation of the nanosorbent (Figure 6), which consists of functionalizing filters with magnesium nanoparties then conducting silanization by passing a polymer solution through the nanoparticle-coated filter. In Phase II, we propose to develop a pilot-scale system for large scale functionalization of 50 x 50 cm sheets of paper to produce hundreds pf nanosorbent disks within a few minutes. The system will be optimized to ensure the homogeneity and reproducibility of the functionalization by studying the geometry of the container, the flow rate and the porosity of the substrate supporting the filter sheets. We will also design and build a die-cutting machine to rapidly cut the nanosorbents into desired disk sizes to be used inside the eBiosampler and the Biospin columns.