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.

Development and Validation of a Panel of One-Step Four-Plex qPCR/RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Pathogens Associated with Canine Infectious Respiratory Disease Complex

“…In view of our data, we have demonstrated that using our assays in a multiplex format did not affect the analytical parameters. Additionally, these parameters were similar to those from other assays recently developed in our laboratory for the detection of canine enteric viruses and feline respiratory pathogens [69,70]. In this study, two to three log 10 improvement of the LOD 95 was determined when compared to the previously published multiplex PCR assays for the detection of CAdV-2, CDV, CIV, and CPiV [38].…”

A Quadruplex Reverse Transcription Quantitative Polymerase Chain Reaction for Detecting Canine Coronavirus, Canine Rotavirus, Canine Parvovirus, and Canine Distemper Virus

“…These positive samples for CCoV, CRV, CPV and CDV had Ct values of 7.68- 35.83, 15.34-35.22, 8.46-35.06, and 9.25-35.11, respectively. The positivity rates of CCoV, CRV, CPV, and CDV in the 1028 clinical samples with the reference assay [24] were 9.05% (93/1028), 0.88% (9/1028), 25.68% (264/1028), and 4.86% (50/1028), respectively.…”

“…The 1028 clinical samples collected from December 2021 to June 2023 were tested by multiplex RT-qPCR, as reported by Thieulent et al [24], for CCoV, CPV, CRV, and/or CDV. The assay described by Thieulent et al [24] used quadruplex RT-qPCR to detect the CCoV ORF5 gene, CPV VP2 gene, CDV Phosphoprotein gene, and canine adenovirus-1 (CAdV-1) Hexon gene, and it used duplex RT-qPCR to detect the CRV NSP3 gene and SARS-CoV-2 N gene.…”

“…The 1028 clinical samples collected from December 2021 to June 2023 were tested by multiplex RT-qPCR, as reported by Thieulent et al [24], for CCoV, CPV, CRV, and/or CDV. The assay described by Thieulent et al [24] used quadruplex RT-qPCR to detect the CCoV ORF5 gene, CPV VP2 gene, CDV Phosphoprotein gene, and canine adenovirus-1 (CAdV-1) Hexon gene, and it used duplex RT-qPCR to detect the CRV NSP3 gene and SARS-CoV-2 N gene. The assay had analytical sensitivity (LOD) of 6 copies/µL (150 copies/reaction) for CCoV, 14 copies/µL (350 copies/reaction) for CPV, 5 copies/µL (125 copies/reaction) for CDV, and 35 copies/µL (875 copies/reaction) for CRV.…”

“…The assay had analytical sensitivity (LOD) of 6 copies/µL (150 copies/reaction) for CCoV, 14 copies/µL (350 copies/reaction) for CPV, 5 copies/µL (125 copies/reaction) for CDV, and 35 copies/µL (875 copies/reaction) for CRV. In our study, these 1028 clinical samples were tested for CCoV, CPV, CDV using triplex RT-qPCR, and were tested for CRV using singleplex RT-qPCR described in the reference assay [24].…”