Rapid identification of SARS-CoV-2 variants using stable high-frequency mutation sites.

Respiratory infectious viruses, including SARS-CoV-2, undergo rapid genetic evolution, resulting in diverse subtypes with complex mutations. Detecting and differentiating these subtypes pose significant challenges in respiratory virus surveillance. To address these challenges, we integrated ARMS-PCR with molecular beacon probes, allowing selective amplification and discrimination of subtypes based on adjacent mutation sites. The method exhibited high specificity and sensitivity, detecting as low as 10 4 copies/mL via direct fluorescence analysis and ~10 6 copies/mL using real-time PCR. Our robust detection approach offers a reliable and efficient solution for monitoring evolving respiratory infections, aiding early diagnosis and control measures. Further research could extend its application to other respiratory viruses and optimize its implementation in clinical settings.