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