Performance evaluation of a SYBR Green-based real-time quantitative PCR for SARS-CoV-2 detection from animal oropharyngeal samples

Abstract

The global emergence of SARS-CoV-2 has highlighted the need for rapid, sensitive, and affordable diagnostic tools, not only for human health but also for animal surveillance within a One Health framework. This study aimed to evaluate the performance of a SYBR Green-based real-time quantitative PCR (qPCR) assay for the detection of SARS-CoV-2 from animal samples, focusing on domestic dogs and cats. A total of 140 oropharyngeal swab samples were collected and analyzed using primers targeting a 139-bp fragment of the N gene of SARS-CoV2. The assay conditions were optimized through gradient PCR, primer concentration adjustment, and melting curve analysis. Cloning and quantification of the target gene allowed the determination of the limit of detection (LOD), which was estimated at 2.1 × 102 copies/µL. Among the samples tested, 13 were positive for SARS-CoV-2, confirmed by a commercial probe-based qPCR. The assay here evaluated demonstrated high specificity, with no cross-reactivity to canine or feline coronaviruses, and had a highly linear standard curve of 0.977 (R² = 0.997) with a value range of quantification cycle (Cq) from 9.25 to 34.89. In addition, it exhibited a 2-log increase in sensitivity compared to conventional PCR. The intra- and inter-assay coefficients of variation were below 1.1 % and 2 %, respectively, confirming high reproducibility. These results support the use of SYBR Green real-time qPCR as a cost-effective and reliable alternative for SARS-CoV-2 detection from animal samples, particularly in resource-limited settings, serving as a tool for epidemiological control of SARS-CoV-2 infection in animal populations.