Multiplex One-Step RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Enteric Viruses of Dogs and Cats

Abstract

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was transmitted from humans to dogs and cats (reverse zoonosis) during the COVID-19 pandemic. SARS-CoV-2 has been detected in fecal samples of infected dogs and cats, indicating potential fecal-oral transmission, environmental contamination, and zoonotic transmission (i.e., spillback). Additionally, gastrointestinal viral infections are prevalent in dogs and cats. In this study, we developed and validated a panel of multiplex one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays for the simultaneous detection of SARS-CoV-2 and common canine enteric viruses: Canine Enteric Assay_1 (CEA_1) for the detection of canine adenovirus-1, canine enteric coronavirus, canine distemper virus, and canine parvovirus, and CEA_2 for the detection of rotavirus A (RVA), and SARS-CoV-2); or common feline enteric viruses (Feline Enteric Assay_1 (FEA_1) for the detection of feline enteric coronavirus, feline panleukopenia virus, RVA, and SARS-CoV-2). All assays demonstrated high analytical sensitivity, detecting as few as 5-35 genome copies/µL in multiplex format. The repeatability and reproducibility of the multiplex assays were excellent, with coefficient of variation <4%. Among the 58 clinical samples tested, 34.5% were positive for at least one of these viruses, and SARS-CoV-2 was detected in two samples collected from one dog and one cat, respectively. In conclusion, these newly developed one-step multiplex RT-qPCR assays allow for rapid diagnosis of enteric viral infections, including SARS-CoV-2, in dogs and cats.

Keywords: SARS-CoV-2; canine enteric virus; diagnostics; feline enteric virus; multiplex; reverse transcription–qPCR (RT-qPCR); reverse zoonosis; zoonosis.

Figure 1

Comparison of analytical sensitivity of each singleplex and multiplex RT-qPCR assays for the detection of SARS-CoV-2 and canine and feline enteric viruses. CEA: Canine enteric assay; Ct: Cycle threshold; FEA: Feline enteric assay; IVT RNA: in vitro transcribed RNA; R²: linearity; E: Efficiency.