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Although antigen test kit (ATK) is used for COVID-19 screening within 15\u201330\u00a0min, the drawback is its limited sensitivity. Hence, a rapid one-step quadruplex real-time RT-PCR assay: termed \u01a9S COVID-19 targeting ORF1ab, ORF3a, and N genes of SARS-CoV-2; and Avocado sunblotch viroid (ASBVd) as an internal control was developed. Based on strategies including designing high melting temperature primers with short amplicons, applying a fast ramp rate, minimizing hold time, and reducing the range between denaturation and annealing\/extension temperatures; the assay could be accomplished within 25\u00a0min. The limit of detection of ORF1ab, ORF3a, and N genes were 1.835, 1.310, and 1 copy\/reaction, respectively. Validation was performed in 205 combined nasopharyngeal and oropharyngeal swabs. The sensitivity, specificity, positive predictive value, and negative predictive value were 92.8%, 100%, 100%, and 97.1%, respectively with 96.7% accuracy. Cohen\u2019s Kappa was 0.93. 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