Performing Department
(N/A)
Non Technical Summary
Severe acute respiratory syndrome coronavirus 2 (SARS?CoV?2) demonstrated the ability to infect many animal species. Widespread natural SARS-CoV-2 infection of white-tailed deer in the USA raised concerns about their role as a reservoir of SARS-CoV-2. As the virus continues to circulate in animals, it is likely to mutate and could threaten the health of agricultural animals. Therefore, it is critical to monitor the natural SARS-CoV-2 infection of animals and the genetic changes in the virus. Current methods for detecting and genetic sequencing of SARS-CoV-2 require expensive instrumentation, are not standardized for animal samples, and are unsuitable for field application. Hence, this project aims to develop and validate highly sensitive, cost-effective, and flexible nanopore-based molecular methods to monitor the circulation of SARS-CoV-2 in agricultural animals. The project will support effective surveillance of SARS-CoV-2 infection of animals through the rapid detection and characterization of SARS-CoV-2.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
This project aims to develop and validate molecular methods for detecting and sequencing SARS-CoV-2 in animal samples. We propose RT-LAMP and rhAmpSeq to detect and sequence SARS-CoV-2 variants, respectively, from farmed animal samples.The three specific aims are below.Aim 1: To develop an RT-LAMP assay for the SARS-CoV-2 detection in animal samples:Aim 2: To develop a rhPCR MinION-based rapid and cost-effective method for sequencing SARS-CoV-2 variants in animals:Aim 3: To validate RT-LAMP and rhPCR Minion Sequencing with RT-PCR and Illumina sequencing.
Project Methods
Aim 1: To develop an RT-LAMP assay for the SARS-CoV-2 detection in animal samples: Further building on our recent successful development of RT-LAMP coupled nanoparticle assay for rapid detection of SARS-CoV-2 from human samples, we will develop a sensitive and flexible RT-LAMP assay for SARS-CoV-2 testing in cattle and farmed deer samples.Aim 2: To develop a rhPCR MinION-based rapid and cost-effective method for sequencing SARS-CoV-2 variants in animals: We will further develop the rhPCR Nanopore method to detect SARS-CoV-2 variants in cattle and farmed deer samples to achieve the resolution of shotgun sequencing with the cost-effectiveness of targeted sequencing (< US$60/sample).Aim 3: To validate RT-LAMP and rhPCR Minion Sequencing with RT-PCR and Illumina sequencing. We will validate the RT-LAMP and rhPCR assays, comparing them with RT-PCR and Illumina sequencing methods to determine analytical sensitivity and specificity using samples spiked with inactivated SARS-CoV-2 virus variants. We will then use a repository of positive and negative clinical samples (human and white-tailed deer) from our collection to assess the diagnostic sensitivity and specificity of RT-LAMP and rhPCR assays compared with RT-PCR and Illumina sequencing.