Recipient Organization
UNIV OF PITTSBURGH
(N/A)
PITTSBURGH,PA 15260
Performing Department
(N/A)
Non Technical Summary
SARS-CoV-2, the virus responsible for COVID-19, has been found in various animal hosts, including white-tailed deer in the USA. These findings raise concerns about the potential transmission of the virus from deer to livestock, particularly cattle. To address this spillover risk, it's crucial to monitor how SARS-CoV-2 evolves in farmed deer and its potential infection of cattle. However, current methods for detecting and sequencing the virus are expensive, not tailored for animal samples, and unsuitable for the field use. This project aims to develop cost-effective and adaptable molecular methods for monitoring SARS-CoV-2 in agricultural animals.The project will develop two methods: 1. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) with solidstatenanopore sensing and 2. an amplicon-based sequencing method rhAmpSeq. This project brings together a diverse team of experts in virology, molecular biology, biomedical engineering, clinical veterinary microbiology, and veterinary diagnostics to develop and rigorously validate these methods. These proposed methods are promising for cost-effective, flexible, and rapid detection and genetic characterization of SARS-CoV-2. This research aligns with the American Rescue Plan (ARP) Surveillance Program's goal of developing surveillance tools for rapidly detecting and characterizing infectious agents like SARS-CoV-2.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
The overarching hypothesis of this project is that sensitive, rapid, and cost-effective nanopore-based methods (RT-LAMP and rhPCR coupled with real-time sequencing) will lead to efficient surveillance of SARS-CoV-2 and its drifted variants in farmed animals.The project aims to develop and validate molecular methods for detecting and sequencing SARS-CoV-2 in animal samples. The two methods proposed are RT-LAMP and rhAmpSeq to detect and sequence SARS-CoV-2 variants, respectively, from farmed animal samples (deer and cattle). We will carry out a robust validation of the methods following the guidelines of the World Organization of Animal Health (WOAH), previously known as the Office International des Epizooties (OIE), and the American Association of Veterinary Laboratory Diagnosticians (AAVLD).The three specific aims are below.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 flexibleRT-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 shotgunsequencing 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 samplesspiked 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-PCRand Illumina sequencing.
Project Methods
Control coronavirus RNA creation: Heat-inactivated SARS-CoV-2 variants will be produced in our ABSL3 laboratory. Non-SARS-CoV-2 animal coronavirus will be heat-inactivated in our BSL2 laboratory. Nucleic acid (RNA) extraction will be performed from inactivated pure viral cultures (SARS-CoV-2 or non-SARS-CoV-2 animal coronaviruses) with an established protocol using a KingFisher (ThermoFisher Scientific) with the MagMAX Viral/Pathogen extraction kit (ThermoFisher Scientific). We have appropriate permits from APHIS, CDC, and Penn State's Institutional Biosafety Committee to handle SARS-CoV-2 viruses in the ABSL3 lab and multiple animal coronaviruses from cattle, pigs, and chickens in the BSL2 lab. We have all the currently known SARS-CoV-2 variants and endemic coronaviruses of cattle, chickens, and pigs in our possession.Preparation of spiked samples: Nasal swab-containing medium spiked with RNA from animal coronaviruses will be used for the optimization of the RT-LAMP assay. The quantity of viral genome copies will be determined by quantitative real-time PCR. RNA will be spiked into nasal swab media at various concentrations ranging from 101 to 105 copies/μl.Nucleic acid extraction: RNA will be extracted from the spiked nasal swab media using a King- Fisher (ThermoFisher Scientific) with the MagMAX Viral/Pathogen extraction kit (ThermoFisher Scientific). Concentration and quality will be assessed with a Qubit and Nanodrop, respectively.SARS-CoV-2 RT-LAMP:Isothermal RT-LAMP amplification will be performed with RNA from spiked samples as the starting material. The total volume of the RT-LAMP assays will contain 24 μl master mix and 1 μl RNA sample. The master mix includes isothermal buffer, PCR grade water, MgSO4 (7 mM), Syto 9 Green (0.5 μM), deoxyribonucleotide triphosphates (dNTPs, 1.4 mM), Bst 2.0 DNA polymerase (0.4 U/μl), Warmstart reverse transcriptase (0.3 U/μl), primer sets (0.2 mM F3 and B3c, 1.6 mM FIP and BIP, 0.8 mM LF and LB). The reactions will be performed at a constant temperature of 65Cusing either a benchtop PCR instrument or a customized heat block. All reactions will have an additional 25 μl mineral oil added to prevent evaporation and cross-contamination.Nanopore sensing and data analysis: A typical voltage of 200 mV will be applied across the nanopore constriction with a 6363 DAQ card (National Instruments, USA). The ionic current traces will be amplified by Axopatch 200B (Molecular Device, USA), low pass filtered at 10 kHz, and digitalized by the 6363 DAQ. The data will be acquired by a customized LabVIEW software (National Instruments, USA). The nanopore measurement system will be placed inside a homemade Faraday cage to shield the environmental noise. A custom-built MATLAB (Math- Works, USA) program will be developed to analyze the current drop, duration time, ECD, and event rate.rhPCR, library preparation, nanopore sequencing, and data processing. Extracted RNA will undergo dual-PCR rhPCR library preparation following manufacturer's instructions. Briefly, rhPCR primers will be combined with rhAmpSeq71 library mix #1 to amplify targets of interest. The rhPCR amplicons from step 1 will be prepared for sequencing with the Oxford NanoporeRapid Barcoding Kit. After barcoding, the samples will be pooled into one library and loaded into the R9 flow cell. Sequencing will be run for up to 12 hours, and data will be processed with EPI2ME72 using the standard SARS-CoV-2 Workflow.