Projects
TETHERED ENZYME TEST FOR HIGHLY ACCURATE, RAPID, FIELD-BASED DETECTION OF AVIAN INFLUENZA
Topic: Influenza
Summary
Non Technical Summary
Highly pathogenic avian influenza (HPAI), already a major problem for poultry farms, has recently caused outbreaks in dairy cattle herds around the country, drastically reducing the viable milk output of affected cows. Furthermore, evidence shows that the virus can be transmitted to humans through contact with infected animals, raising concerns of increased spread and possible recombination with a human strain of influenza. The USDA has implemented mandatory testing for animals before crossing state lines, but testing beyond that remains extremely limited due to the turnaround time associated with the current gold standard method, RT-PCR, which requires samples to be shipped to a laboratory. A rapid, on-site test is required for farmers to make meaningful management decisions regarding the sequestering of sick animals. To address this need, TETmedical is developing a sensitive, rapid, PoC diagnostic test for HPAI H5N1 using their innovative tethered enzyme technology (TET) platform. This technology utilizes enzymes which are tethered to nanoparticles to generate a rapid viral RNA biosensor with a bioluminescent readout. The oriented immobilization of the enzymes on the nanoparticles confers advantages in speed, specificity, and sensitivity. Here, we propose to utilize sequence-specific identification oligonucleotides to target the enzymatic reaction to H5N1 RNA. A similar TET test for SARS CoV-2 RNA demonstrated high sensitivity and a 5-minute reaction time. The key advantage of this method over existing diagnostic techniques is the specificity and rapid turnaround time enabled by on-site testing, which enables immediate interventions that can reduce the spread within and between farms.
Objectives & Deliverables
Goals / Objectives
TETmedical is developing a novel testing platform based on tethered enzyme technology (TET) to meet the rising need for rapid and highly sensitive point-of-care (PoC) diagnostics. Recent outbreaks of highly pathogenic avian influenza (HPAI) in flocks of chickens and herds of dairy cattle across the country have led to calls for increased diagnostic testing of both animals and humans to control the spread of the virus. In addition to bird-to-bird transmission, the virus is capable of mammal-to-mammal transmission, and as such poses a serious threat to poultry and dairy farms everywhere, as well as humans should it gain a foothold in the human population. In Phase I of this SBIR project, we propose to develop a highly sensitive, highly specific, and rapid, diagnostic test for HPAI H5N1 using our innovative TET platform. The first version of the test will significantly improve throughput in diagnostic labs, reducing test time from several hours to <10 minutes. Phase I will provide an essential foundation for Phase II, which would target development of a field (PoC) test to provide immediate results. The proposed work addresses the USDA Research Priority for the development of marketable technologies designed for use in agriculturally important animals that will (1) improve the safety and/or quality of end products derived from animals, (2) improve animal health and well-being, and (3) improve production efficiency.This will be achieved through the following Phase I Objectives: Objective 1: Develop a rapid (<10 min) molecular assay for specific detection of HPAI H5N1 RNA Objective 2: Validate detection of H5N1 RNA from field-collected samples
Challenges
Project Methods
TETmedical's proposed RapidNA (Rapid Nucleic Acid) test is designed to facilitate rapid on-site testing of poultry, eggs, cows, milk, and humans to mitigate the spread of the virus at and between farms and to improve overall animal health, human health, and milk production and quality. The key advantage of this method over existing diagnostic technologies is the combination of highly specific and highly sensitive molecular testing and rapid turnaround time enabled by on-site testing. This combination facilitates immediate management decisions and interventions that can reduce the spread within and between farms.To develop theRapidNA technology, this project will include several key R&D methods:1. enzyme production- TETmedical uses a combination of molecular and chemical approaches to produce, purify, isolate and analyze its TET enzymes for use in this rapid molecular test.2. enzyme-basedassay development- including testing and analysis kinetic data from enzyme and substrate mixtures, calculations of luminescence kinetics, kinetic data analysis, and statistical analysis3. bioinformatics – computational tools to fine-tune assay specificity and sensitivity via engineering of optimal ID-Oligos4.virus and RNA methods- including virus isolation, RNA extraction and quantification techniques 5. product analytical testing – including side-by-side validation of the RapidNA against the gold-standard RT-PCR.
