Non Technical Summary
Bovine viral diarrhea virus (BVDV) is a significant cause of morbidity in beef cattle and costs the U.S. beef cattle industry $1.5 to 2.5 billion annually. BVDV decreases herd productivity and increases beef prices for consumers. It is therefore necessary to manage and ideally eradicate BVDV in beef cattle herds. Screening entire herds for the virus is necessary for eradication, especially since cattle can be persistently infected with BVDV and show minimal or no symptoms. However, there are no commercialized BVDV tests that are rapid, user-friendly, and capable of mass screening.Our project's goal is to innovate a diagnostic kit called SpinTest BVDV, which enables simultaneous testing of over one hundred cattle with high accuracy in less than an hour. Our core technology is a cutting-edge, lab-on-a-chip which would enable any rancher or veterinarian to perform advanced diagnostics on the farm with easy-to-read results. SpinTest BVDV will be commercialized: it will decrease disease screening workloads and costs for cattle ranchers, improve cattle productivity, and promote animal well-being. This equates to higher quality beef at lower prices for U.S. consumers and improved U.S. agricultural security.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3310	1040	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1040 - Molecular biology;

Goals / Objectives
Our project's over-arching goal is to develop a novel veterinary diagnostic technology for beef cattle, specifically for mass screening of bovine viral diarrhea virus (BVDV). Cattle ranchers and livestock veterinarians need to screen large numbers of cattle for BVDV, otherwise it can can severely undermine herd health and productivity. Current diagnostic methods are inadequate for the needs of the beef cattle industry. Although tests at centralized laboratories can pool bovine samples for higher throughput, these samples must be shipped to the laboratory, incurring long turnarounds. Pen-side tests can yield results within an hour but cannot utilize pooled samples. This means animals must be individually tested by limited ranch personnel, making screening entire herds expensive and tedious. Eradicating BVDV in the United States would greatly benefit cattle herds, but this is unfeasible with current technologies. Our proposed solution, SpinTest BVDV, would streamline BVDV surveillance for cattle producers and help accomplish and maintain eradication.The following objectives are integral to developing our proposed SpinTest BVDV product and achieving our goals:Objective 1 - Showing that minimally processed bovine ear notch samples are usable for BVDV detection via reverse transcription loop-mediated isothermal amplification (RT-LAMP). Any sample processing required should be workable at pen-side with minimal training and effort to make our test more user-friendly.Objective 2 - Showing that RT-LAMP is sensitive enough to detect BVDV in pooled samples. Larger possible pool sizes provide flexibility to the end-user and can help save time and effort when screening large numbers of cattle.Objective 3 - Demonstrating that pooled RT-LAMP assays for BVDV can be accurately assessed visually using centrifugal microfluidics in combination with a spincubator apparatus. Color readouts that can be assessed by eye will simplify the design of the spincubator (i.e., the device that actuates and incubates the SpinTest BVDV centrifugal microfluidic chips).By this project's completion, we will have developed a functional prototype which can then be refined into a product ready for commercialization in subsequent projects.

Project Methods
Our project comprises four stages, each with a characteristic set of methods:Stage 1: Ensuring Sample Compatibility with RT-LAMPLoop-mediated isothermal amplification (LAMP) and, more specifically, reverse transcription LAMP (RT-LAMP) are the basis of our BVDV testing method. We will first verify RT-LAMP compatibility with bovine samples by assessing abnormalities in LAMP and RT-LAMP amplification. We will utilize ear notches for BVDV testing since they are commonly used by ranchers and livestock veterinarians for currently available BVDV tests. Viral particles will be extracted by incubating ear tissue in a buffer and syringe filtering the extract to remove contaminants.For LAMP and RT-LAMP experiments, we will utilize the LavaLAMP polymerase, which amplifies both DNA and RNA templates. We will assess LAMP using a primer set targeting M13mp18 ssDNA and determine if the reactions maintain sensitivity and specificity in the presence of bovine ear notch extract. Reaction readouts will be mediated by methyl green (MeG) dye: positive reactions will appear visibly greenish-blue whereas negative reactions will be colorless. The M13mp18 ssDNA will be serially diluted in water and the 95% limit-of-detection (LOD95) quantified via probit analysis in R. To determine if ear notch extracts cause inhibition, the extracts will be spiked with an LOD95-quantity of M13mp18 ssDNA and then added to LAMP reactions; the reactions will be 60% extract by volume. The proportion of positive reactions will be calculated for assays with and without the ear notch extract. No-template control (NTC) reactions will also be performed, to determine if extracts cause spurious amplification. If inhibition (greater than 50% reduction in positivity at LOD95) or spurious amplification (over 5% positivity for NTC reactions) is not observed, then we will perform a similar assessment with control RT-LAMP reactions using primers designed against E. coli bacteriophage MS2 RNA.Stage 2: BVDV Primer Design and SelectionWe aim to develop a test capable of detecting all BVDV strains. Genomic data can be accessed via GenBank, which has a compendium of complete/near-complete genomes for BVDV-1 and BVDV-2. This data will be compiled and used for LAMP primer design with PrimerExplorer V5. We will test at least three LAMP primer sets to ensure satisfactory performance. The SpinTest BVDV product will also include internal controls(i.e., LAMP primers designed against endogenous bovine RNase P or 18S ribosomal RNA) to indicate if reactions are incubated properly.The primer sets will be first evaluated using synthetic gene fragments corresponding to their respective targets. High fragment copy numbers (i.e., 10,000 copies per LAMP reaction) will be used for initial screening. Primer sets which enable amplification within an hour at typical concentrations as assessed by endpoint MeG readouts will be further tested. We will ensure that the primer sets do not elicit spurious amplification within the same timeframe using NTC reactions and select the one which yields the shortest time to result. This is all done in microtube format on a heat block. For RT-LAMP evaluation, BVDV RNA controls and samples obtained from persistently infected (PI) animals will be used. PI tissue sample extracts will serve as positive controls and when used will comprise 60% of RT-LAMP reaction volumes. We will conduct a similar set of assessments as with the synthetic gene fragments.Stage 3: Integration of RT-LAMP into Centrifugal MicrofluidicsOnce a lead primer set is selected, the RT-LAMP reagents can be integrated into microfluidic chips. The microfluidic chip will feature five identical microfluidic branches. Each branch will have five reaction chambers: one negative control without primers, one positive control for endogenous bovine RNA, and three replicate BVDV reactions. The chips will be fabricated by CNC milling of transparent plastic and sealed using a die-cut base laminated with an adhesive tape. Sample chambers on the chips will be filled via an inlet using a pipette. The chips will then be actuated by spinning at fixed frequencies. The actuation and subsequent incubation of the chip will take place in a device we call a spincubator. The spincubator will include a camera module and can interface with a personal computer. It will perform real-time colorimetry for RT-LAMP experiments and streamline optimization.The microfluidic chips will incorporate lyophilized enzymes and reagents for improved shelf life. The LavaLAMP enzyme and associated reagents are formulated for lyophilization compatibility. This will facilitate in situ lyophilization within the chip's reaction chambers. RT-LAMP reaction composition will be optimized based on lyophilized enzymes to decrease time-to-results for positive reactions and delay false positives in NTCs. Starting with the manufacturer's reaction composition, reagents will be adjusted to improve performance. The concentrations of Mg2+, dNTPs, primers and betaine will be optimized through single factor and orthogonal experimentation. Incubation temperature will similarly be optimized. Real-time colorimetric measurements from the spincubator imaging rig will quantify the benefit of each modification.With the optimized RT-LAMP chemistry, positive PI samples will be diluted with healthy samples to assess maximum pooling. Dilutions of 1:10, 1:20, 1:30, 1:50 will be assessed and the 99% limit-of-detection (LOD99) quantified. We will then determine optimal cutoff times for judging reaction outcomes. Real-time data will be collected for NTC RT-LAMP reactions or reactions with an LOD99 level of PI sample. A receiver operating characteristic (ROC) curve will be generated based on varying cutoff times and the time which maximizes the Youden's index will be chosen.Stage 4: Evaluation of Multi-Pool Testing on Centrifugal MicrofluidicsEach microfluidic branch on a chip can assay a pooled sample; with 5 branches per chip, this entails 5 pools per chip, enabling multi-pooled diagnostics. To evaluate multi-pooling, samples from at least 25 healthy animals will be obtained and mixed in different combinations. We will use at least five chips (equivalent to 25 separate branch tests) for both quantifying sensitivity and specificity in pools with and without a sample from a PI animal, respectively. The samples mixed into pools will be combined in varying combinations and proportions. Assuming there are 25 healthy animals and one PI animal and that pool sizes of 25 are feasible, two sets of experiments would be performed. For negative control experiments, five sample pools will be generated: one with all 25 healthy samples mixed, and four with different combinations of 6 healthy samples mixed. In each pool, each sample is equally represented. The differing combinations elucidate differential impacts of individual sample composition on on-chip RT-LAMP reaction performance. Similarly, 5 pools can be made with the PI sample for positive control experiments where it will constitute 1 part in 25 of the pools. Thus, the influence of variability in pooled sample composition can be assessed.Chips will be incubated for the optimal incubation time and then assessed visually by three individuals. If 2 or 3 BVDV reactions are positive in a branch, then the branch will be considered positive. Otherwise, it will be considered negative. Using this criterion, we will evaluate sensitivity and specificity. Given that pool sizes are set such that positive samples are diluted to LOD99 levels for individual reactions, we expect greater than 99% sensitivity for the on-chip assays. Since the reaction chambers produce independent binary outcomes, 99% sensitivity for a single reaction equates to 99.97% sensitivity for a microfluidic branch. We expect practically no false positives since specificity is similarly enhanced with replicate reactions.