Recipient Organization
NUCLEIC SENSING SYSTEMS LLC
1865 ASHLAND AVE
SAINT PAUL,MN 551045949
Performing Department
(N/A)
Non Technical Summary
As aquaculture increases productivity to meet worldwide demand, it faces an increasing threat from thespread of diseases and pathogens. However, biosensor technologies have not kept pace with the need to monitor these ever-increasing threats. In particular, biomonitoring has been limited by the need for continuous human presence, whether at the point of collection or in the laboratory. What is needed are biosensors that function as robustly and easily as a smoke-alarm, i.e., autonomous, reliable, in-field monitoring technologies capable of disseminating data straightforwardly and in real-time to a broad range of personnel and decision-makers.NS2 will seek to overcome these barriers. By completing the assembly of a benchtop submodule and verifying its efficacy to detect target viral pathogens (Infectious Hematopoietic Necrosis Virus, Viral Hemorrhagic Septicemia Virus, Infectious Pancreatic Necrosis Virus), we will validate and develop the technology. Simultaneously, we will be using the engineering and application data gathered from the submodule to inform an Alpha Product unit's professional industrial engineering design suitable for production and sales.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
Nucleic Sensing Systems will work in partnership with Dr. Jerri Bartholomew at the Oregon State University's John L. Fryer Aquatic Animal Health Laboratory and the Oregon Department of Fish and Wildlife's Fish Health Services to develop and validate the efficacy of a continuous, autonomous, viral detection instrument by adding reverse transcription (RT) to the Tracker's internal process flow. This work will build upon our work funded by NOAA SBIR I & II for the simultaneous eDNA detection of multiple waterborne infectious organisms in aquaculture.Technical Question: What design modifications are necessary to adapt the current eDNA Tracker to an eRNA Tracker?Technical Objectives:Validate RT-qPCR kits on reference eRNA.Incorporate on the information gathered from the RT-qPCR kit development into the design features of the RT-qPCR benchtop prototype.Demonstrate and validate viral detection capabilities of the prototype with the following fish viruses: Infectious Hematopoietic Necrosis Virus (IHNV), Viral Hemorrhagic Septicemia Virus (VHSV), Infectious Pancreatic Necrosis Virus (IPNV). Combined, these viral targets account for the majority of pathogenic losses in the salmon aquaculture industry. Furthermore, both the Oregon Department of Fish and Wildlife as well as Oregon State University identified these viruses as critical targets.
Project Methods
Technical Objective 1: Validate RT-qPCR kits on reference eRNA.Reverse Transcription (RT) enzymes vary by kit and not all are compatible with the Tracker's sample processing methods. In particular, the robustness of the Tracker relies on the lack of a need for introducing or removing reagents during processing beyond a single sample introduction event. This limits our selection of available RT-qPCR kits to "one-step" type kits. Such one-step RT kits typically function at a fixed elevated temperature of about 95degC. We need to ensure that the selected kits function with the Tracker's hot-start MasterMix (the polymerase, free nucleotides, and other buffer reagents), which, while commercial and off the shelf, is specific to droplet digital quantification. Kits to test include: Thermo Fisher TaqPath 1-Step RT-QPCR Master Mix; Takara 1-step RT-qPCR kit; Qiagen OneStep RT-PCR kit; One-Step RT-ddPCR Advanced Kit (BioRad); AgPath-ID™ One-Step RT-PCR Kit (Life Technologies, CA, USA)Stocks of the three target viruses are maintained by ODFW and will be inoculated onto a layer of visually healthy CHSE-214 cells (LaPatra et al. 2003, ODFW SOP ref). Inoculated cultures will be incubated at 15°C and observed for cytopathic effect (CPE). Virus will be harvested when total CPE is observed, centrifuged for 15 min and stored in aliquots at -80°C. These stocks will be used for all subsequent experiments.One aliquot from each virus stock will be used to determine virus concentration by standard plaque assay methods (LaPatra et al. 2003). IHNV and VHSV plaque assays will be performed on EPC cells; IPNV plaque assays will be performed on CHSE-214 cells. Inoculated cell culture plates will be overlaid with methyl cellulose and incubated for 14 days to observe cytopathic effects and then stained with crystal violet - formalin. Plaques will then be enumerated and reported as plaque forming units per mL (pfu/mL.).The efficacy of the three kits will be tested first on IHNV as we have the most experience with qPCR assays for that virus. The kits will be selected based on their ability to utilize BioRad SuperMix, as so will include the BioRad One-Step RT-ddPCR Advanced Kit. An aliquot of the virus will be thawed and diluted to 1010 pfu/mL. Further 10-fold dilutions will be prepared down to 100 pfu/mL for each virus to determine the limits of detection for each kit. Published virus-specific primers and probes for IHNV will be used (Purcell et al. 2013), and each enzyme kit will be tested on the full dilution series in replicates of five using the Tracker's hot-start MasterMix. A no template control will be included on each plate as a negative control. A positive control will be a replicate series of the dilutions using the published RT-qPCR assay, Reactions will be will be analyzed with a QuantStudio 3 Applied Biosystems thermocycler and in continuous-flow using the simple RT-module provided for assay development by the engineering team as described in Task 2.1 of this proposal. Quantification cycle values will be examined to determine which kit(s) had the highest sensitivity and efficiency, and these will be tested using published protocols on IPNV (Vázquez et al. 2016) and VHSV (Garver et al. 2011) .Technical Objective 2: Incorporate the information gathered from the RT-qPCR kit development into the design features of the RT-qPCR benchtop prototype.Once a droplet digital reagent-compatible kit has been identified, the specific protocols necessary for its function will be incorporated into the Tracker. A parallel fluid pathway has already been included in the current Tracker to handle the additional thermal profile of RT enzymes but this pathway has not yet been used. To complete the fluid pathway and make it operational, we need to specify the thermal profile in the RT enzyme protocol identified in Technical Objective 1.Technical Objective 3: Demonstrate and validate viral detection capabilities of the prototype.Testing the viral detection capabilities of the prototype will be done at OSU. Three aliquots of each virus will be thawed and serial dilutions made in 1L of environmental water to give final dilutions of 108 pfu/mL down to 100 pfu/mL. Dilutions will be held at 4? until run through the Tracker. Positive controls will be the virus dilution series in pure water analyzed by the Tracker, and an equivalent dilution series assayed using standard benchtop qPCR protocols described in Objective 1. Qualification cycles will be analyzed to determine sensitivity and efficiency compared with the control.