Establishment of a Padlock Probe-Based Fluorescence Quantitative PCR Method for the Detection of the SARS-CoV-2...

This study aimed to establish and validate a novel detection method that combines padlock probe technology with fluorescence quantitative PCR (qPCR) for the rapid and specific detection of the SARS-CoV-2 Omicron S371L mutation in the spike protein. Padlock probes and amplification primers were designed and synthesized based on the Omicron S371L mutation site. The probe was designed to anneal with high specificity to the mutation, allowing ligase-mediated circularization only in the presence of the target sequence. The circularized probe then served as a template for qPCR amplification. Assay optimization included probe concentration, ligation temperature, ligase concentration, and ligation time. Analytical sensitivity, specificity, and recovery performance were systematically evaluated. Finally, the method was validated using 30 clinical samples, with results compared to Sanger sequencing. The optimal assay conditions were identified as a padlock probe concentration of 10 nM, ligase at 0.2 U/µL, ligation carried out at 65 °C for 30 min, and 10 µL of the ligated product used for subsequent qPCR amplification. Under these parameters, the assay achieved a limit of detection of 5.78 fM and demonstrated strong linearity (R² = 0.9669). Specificity testing showed clear differentiation between the mutant template and single-, double-, or triple-base mismatches. In performance evaluations, recovery rates ranged from 89.7% to 108.0% in both PBS and urine samples. For clinical validation, the method showed complete agreement with Sanger sequencing results, yielding positive and negative predictive values of 100%. In summary, the padlock probe-based qPCR assay developed in this study offers a fast and reliable approach for detecting the Omicron S371L mutation in SARS-CoV-2. Because the method bypasses the reverse transcription step that is typically required for RNA targets, it simplifies the workflow without compromising accuracy. These features suggest that the assay could be well suited for broader clinical application, particularly in large-scale screening of emerging SARS-CoV-2 variants.