Innovation
Focusing on the research and development and application of isothermal nucleic acid amplification technology
Establishment and Evaluation of a Fluorescent RT-RAA Assay for the Detection of Novel Coronavirus Nucleic Acid
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Abstract: Objective To establish a fluorescent RTRAA-based rapid detection method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods Using in vitro-transcribed SARS-CoV-2 RNA as the template, single-stranded DNA-binding protein, recombinase, and DNA polymerase were employed to rapidly amplify the ORF7ab gene and S-gene fragments at a constant temperature of 40.5°C. The method was preliminarily evaluated using nasopharyngeal swab specimens from confirmed COVID-19 positive patients and other patients with respiratory viral infections. Results The method developed in this study takes less than 20 minutes to detect both SARS-CoV-2 genes. The sensitivity for both genes is 2 copies per reaction tube, and the specificity is 100%. The minimum detection limits of the two primers showed consistent amplification onset times and nearly identical curve profiles in three replicate experiments. Conclusion The method established in this study exhibits high sensitivity, strong specificity, good reproducibility, and high concordance with clinical specimen testing results. Moreover, it does not require expensive equipment, making it suitable for rapid on-site detection.
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