DEVELOPMENT OF RAPID, FLEXIBLE, AND COST-EFFECTIVE MOLECULAR DIAGNOSTIC METHODS FOR COVID-19 DETECTION IN ANIMALS

<b>Project Methods</b><br> 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.