Multi-lab validation of a real-time PCR assay for detection and surveillance of SARS-CoV-2 for companion animal applications.

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Project Summary Following its emergence in 2019, SARS-CoV-2 spread globally, leading to over 757 million confirmed cases of COVID-19 and over 6.8 million deaths worldwide (WHO COVID19 dashboard, as of Feb 27, 2023). Variants of concern (VOC) have emerged throughout the pandemic (CDC, 2021a). Among the recent of these are Delta, which has been associated with increased pathogenicity, and Omicron and its currently circulating subtypes, which are associated with increased transmissibility (CDC, 2021a). Although humans have served as the primary SARS-CoV-2 reservoir, natural infections have also been reported in animals (Singla et al., 2020), mostly either companion animals, including cats and dogs, or other animals under human care, including tigers, lions, gorillas, snow leopards, otters and spotted hyenas (USDA-APHIS). From August 2021 to present, all confirmed cases of SARS-CoV-2 in companion and exotics species in the U.S have been associated with either Delta or Omicron, which also continue to represent a majority of overall infections in these animal populations since the beginning of the pandemic (USDA-APHIS). Accurate detection and differentiation of major VOCs is important for surveillance of SARS-CoV-2 in these animal populations, and can serve as a useful tool for retrospective studies that may help determine the roles these species played during the pandemic. We have previously developed a multiplex real-time PCR (qPCR) assay for the detection of SARS- CoV-2 and differentiation of Delta and Omicron variants (Tsui et al., 2022). Our objective is to perform a multi- lab validation of the current assay, or an updated assay, and to make the assay publicly available for surveillance and diagnostic research of SARS-CoV-2 in current and pandemic-era animal submissions, respectively. Specific proposal objectives are as follows: 1. In silico re-analysis of SARS-CoV-2 genome sequences to ensure the assay is detecting the currently circulating Omicron sub-variants while still maintaining detection and differentiation of Delta variant. 2. Develop a sample panel for multi-lab validation by creating a SARS-CoV-2-negative canine nasal swab pool matrix that will be spiked, at different concentrations, with SARS-CoV-2 positive animal samples. Synthesized target RNA will be used only if positive animal samples are not available. Absolute concentration of each dilution will be measured by digital PCR, and compared to qPCR testing. 3. Distribute the validation sample panel to collaborating laboratories for multi-lab validations. Testing materials for the panel will include RNA extraction reagents, primers, probes, qPCR reaction reagents and positive controls. 4. Real-time PCR data from each collaborative laboratory will be collected, analyzed for inter-lab performance, then summarized for conference presentation and drafted into a manuscript for publication.