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 techniques in aquaculture environments. Current sampling tools still use nitrocellulose filters that are not designed for that purpose. As a result, the sampling effort is significant, and the collected samples contain low eDNA concentrations and a high amount of PCR inhibitors, resulting in unreliable disease detection. In this SBIR Phase I proposal, we offer the demonstration of thefeasibility and proof of concept of a novel eDNA sorbent kit that relies on chemical affinity-based eDNA sorption rather than size exclusion used by conventional techniques. This new concept would allow faster, more efficient, and cost-effective collection and concentration of clean eDNA from large volumes of aquaculture water, thus enabling more reliable and affordable aquatic disease diagnostics. The novel sorbent can also be used to monitor the presence and abundance of farmed fish species but also the presence of aquatic invasive species by a simple eDNA analysis, thus enabling better management of sustainable and productive fisheries and aquaculture, andimproving food safety and security.
Animal Health Component
50%
Research Effort Categories
Basic
20%
Applied
50%
Developmental
30%
Goals / Objectives
This SBIR Phase I proposal aims at demonstrating the feasibility and proof of concept of the first environmental DNA (eDNA) nanosorbent technology for the collection, concentration, and recovery of aquatic eDNA from pathogenic microorganisms to enable more efficient, reliable, and cost-effective disease monitoring in aquaculture environments. The technology would significantly enable and empower management programs in the aquaculture industry, fisheries, invasive species detection, and aquatic ecosystem surveys in the United States.The proposed eDNA aquatic sampling technology will be developed and tested in the following three major steps to answer the question: "Can a nanosorbent specifically and efficiently capture viral and microbial eDNA in aquaculture environments?Specific aim 1: To develop a functional nanosorbent with a high affinity to eDNA,Specific aim 2: To test the efficiency of the developed eDNA nanosorbents,Specific aim 3: To test the feasibility of the new nanosorbent for the capture and recovery of microbial eDNA in real-world samples,
Project Methods
Task 1: Development of the eDNA nanosorbentNanoparticles have been demonstrated to have high affinity to DNA,20-24 while exhibiting antimicrobial properties to prevent biofouling,25,26 which is expected to enhance eDNA collection and preservation. In order to use TiO2, MgO, or APTES nanoparticles for eDNA capture, the nanoparticles need to be integrated into a filter or membrane with pore size ranging from 3 µm to 20 µm to allow large water flow rates without clogging. Frontline Biotechnologies has recently developed a new technology that enables the growth of any metal nanoparticles on various porous substrates, including fabrics, polymers, and filters. The technology has so far been used to grow selenium, iron, copper, and 12 other nanoparticles on polyurethane sponges, polymers, and other porous materials to capture different pollutants from water. Two patents have been filed. In this project, we will initiate and optimize the growth of a number of different nanoparticles including TiO2, MgO, AlO2, or APTES nanoparticles on cellulose and nylon membranes, as those particles are expected to have a high affinity for DNA and exhibit a high pKa. The structure and surface coverage will be analyzed by scanning electron microscopy, X-ray diffraction, and FTIR spectroscopy available at the University of Minnesota Characterization Facility. The functionality of the nanosorbent will be tested by passing DNA solutions through it and quantifying the DNA content before and after filtration using a Qubit fluorometer 3.3 and qPCR available in the co-PI's laboratory. Also, the efficiency of the nanosorbent will be compared to the same filter without nanoparticles to assess the role of the nanoparticles in the eDNA capture.Task 2: Study of the efficiency of the eDNA nanosorbentDNA recovery: After water filtration, the nanosorbent will be immersed in a preservation buffer and stored in sterile conditions at -20 °C in the dark until used (Figure 1). The DNA will be retrieved from the filter using a recovery solution designed to break the bond between the nanoparticles and the eDNA without damaging the genetic material. In the event that the free eDNA is present at low amounts in the environment, we will use a recently developed method by our lab to extract eDNA from collected tissues.31 Briefly, the method involves adding sodium hydroxide pellet to the water sample, causing a sudden increase in temperature. The high temperature combined with the effect of surfactant results in DNA release into the solution within a few minutes. eDNA amplification and quantification: Quantitative PCR (qPCR) will be performed using StepOnePlus Real-Time PCR System available in the Co-PI's laboratory 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 a high filtered volume of water and thus high DNA content, specificity is very important to prevent false negatives in the detection of invasive species. The qPCR assays for the selected species will be based on protocols reported in the literature with some modifications, using best practices and controls necessary for highly sensitive eDNA work. Task 3: Field and Aquarium TestingSampling and filtration: Our approach to evaluating our nanosorbent kit will be to collect simultaneously multiple samples from the same site using a conventional approach and our sampling kit (Frontline's eDNA kit). 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, then sample soon after to create a challenging low eDNA scenario. This initial evaluation is intended as a "proof-of-concept" for the effectiveness of the new nanosorbent, as it is impossible to evaluate the myriad combinations of species, sampling approaches, and environments for which eDNA sampling has been applied. The Minnesota Department of Natural Resources (MN-DNR) and local farm managers will assist with the collection. We will collect the eDNA from aquaculture farms and will detect one from each category of the pathogenic species. The primers will be designed for ITS region for each species and qPCR will be performed. We will compare our eDNA nanosorbent kit to a traditional approach used in many eDNA studies including the filter capsules.34 Each sample is filtered, and eDNA is extracted from the filter and analyzed separately. Data collection and analysis: Field tests will be evaluated by comparisons of mean eDNA copy number per sample and proportion of samples above qPCR limits of detection (i.e, the 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 and varying criteria for the number of replicates that need to be positive to declare the sample positive.35 We will further compare our detection probabilities based on criteria of 1, 2, or all 3 replicates being positive.Task 4: Reporting and result disseminationNo experimental work will be conducted on this task. The focus here will be on results dissemination and reporting. Assessment will be provided by the USDA after receiving the project final report and product samples