Development of multiplex real-time PCR assays for differentiating SARS-CoV-2 from other respiratory and enteric pathogens

PROJECT SUMMARY/ABSTRACT There is an urgent need for developing and validating new and improved molecular and serological tests for the diagnosis of SARS-CoV-2 in companion animals (i.e., dogs and cats). It has been shown that people with COVID-19 frequently pass SARS-CoV-2 on to their pets. Cats are more susceptible to SARS-CoV-2 than dogs, and there is evidence that both species can develop COVID-19 after a natural or experimental exposure. The clinical signs of COVID-19 in cats and dogs include respiratory signs and diarrhea, which must be differentiated from feline respiratory disease complex (FRDC), canine infectious respiratory disease complex (CIRDC or commonly known as "kennel cough"), and diarrhea caused by other viruses, bacterial and parasites. Thus, rapid detection and differentiation of SARS-CoV-2 from other common viral and bacterial agents in a single specimen is critical in controlling COVID-19 and implementing appropriate public health measures. Similarly, there is an urgent need for developing highly specific and sensitive antibody detection (serological) assays to answer fundamental questions about the epidemiology and immune response to SARS-CoV-2 and aid in the implementation and evaluation of control programs. Thus, it is hypothesized that current laboratory testing for COVID- 19 in dogs and cats could be improved by developing new multiplex real-time qPCR/RT-qPCR assays for the detection and differentiation of SARS-CoV-2 in respiratory (nasal and nasopharyngeal) and fecal specimens, as well as developing a sandwich ELISA for the detection of IgM and IgG immune responses using recombinant viral proteins. To test this central hypothesis, we have two specific aims: Aim 1). Develop Multiplex Real-time PCR Assays to Detect SARS-CoV-2 and Other Respiratory and Enteric Pathogens in Dogs and Cats: The objective is to develop a series of multiplex (quadruplex) TaqMan® real-time qPCR/RT-qPCR assays using a combination of fluorescently labeled probes. All assays will be optimized using the new TaqPath 1-Step Multiplex Master Mix; and Aim 2). Develop Sandwich ELISA for the Detection of Antibodies to SARS-CoV-2 in Dog and Cat Serum Samples: To detect IgM and IgG immune response to SARS-CoV-2 in dogs and cats using recombinant nucleocapsid (N) and spike (S) proteins. In summary, multiplex real-time qPCR/RT-qPCR assays are highly sensitive and specific and will rapidly identify and distinguish dogs and cats infected with SARS-CoV-2. The development and validation of both IgM and IgG ELISA will allow identifying animals exposed to the virus. These newly developed assays will enhance the molecular and serologic diagnosis of SARS-CoV-2 in pets, and also, advance epidemiological investigations.