Tailored Multiplex Real-Time RT-PCR with Species-Specific Internal Positive Controls for Detecting SARS-CoV-2 in Canine and Feline Clinical Samples

SIMPLE SUMMARY: Given that SARS-CoV-2 infections in companion dogs and cats have been frequently reported worldwide during the ongoing COVID-19 pandemic, a multiplex real-time RT-PCR assay is urgently required to reliably detect SARS-CoV-2 infection in companion animals. In this study, we developed a tailored multiplex real-time RT-PCR assay to simultaneously detect RdRp and N genes of currently circulating SARS-CoV-2 variants and canine or feline 16S rRNA as an endogenous internal positive control. The developed assay had high sensitivity, specificity, and accuracy and could detect all tested SARS-CoV-2 variants, including Omicron subvariants. Clinical evaluation of canine and feline specimens revealed that the diagnostic sensitivity of the assay was equivalent to that of a commercial SARS-CoV-2 multiplex real-time RT-PCR kit. Furthermore, canine or feline endogenous internal positive control was amplified using the developed assay while avoiding false-negative results. Considering the high sensitivity, specificity, accuracy, and reliability, the developed assay can help diagnose COVID-19 in dogs and cats and potentially play a vital role in the rapid diagnosis and control of SARS-CoV-2 infections in companion animals. ABSTRACT: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been frequently reported in companion dogs and cats worldwide during the ongoing coronavirus disease. However, RT-qPCR methods developed for humans have been used for the diagnosis of SARS-CoV-2 infections in suspected companion dogs and cats owing to the lack of the companion animal-tailored methods. Therefore, we developed a multiplex RT-qPCR (mRT-qPCR) using newly designed primers and probes targeting RdRp and N genes of all currently circulating SARS-CoV-2 variants as well as the canine or feline 16S rRNA gene as an endogenous internal positive control (EIPC) for reliable diagnosis of SARS-CoV-2 infection from suspected dogs and cats. The developed mRT-qPCR assay specifically detected the target genes of SARS-CoV-2 but no other canine or feline pathogens. Furthermore, canine and feline EIPCs were stably amplified by mRT-qPCR in samples containing canine- or feline-origin cellular materials. This assay has high repeatability and reproducibility, with an optimal limit of detection (<10 RNA copies per reaction) and coefficients of variation (<1.0%). The detection rate of SARS-CoV-2 of the developed mRT-qPCR was 6.6% for canine and feline nasopharyngeal samples, which was consistent with that of a commercial mRT-qPCR kit for humans. Collectively, the newly developed mRT-qPCR with canine and feline EIPC can efficiently diagnose and evaluate the viral load in field specimens and will be a valuable tool for etiological diagnosis, epidemiological study, and controlling SARS-CoV-2 infections in canine and feline populations.