Abstract
SARS-CoV-2 variants of concern (VOCs) emerged in late 2020 and were characterized by increased transmissibility, virulence, or antigenic drift. Rapid identification in diagnostic materials is essential for effective implementation of public health responses. The spike gene, essential in viral entry and immune escape, frequently harbors VOC-specific mutations. Their detection by conventional sequencing methods remains time-consuming. A nested RT-PCR-based pyrosequencing (PSQ) assay was designed targeting three mutation-dense regions of the SARS-CoV-2 spike gene. Analytical specificity was assessed using defined controls. Clinical validation involved 175 SARS-CoV-2-positive specimens from the national German acute respiratory infections (ARI) sentinel covering Alpha, Beta, and Delta variants, with different subsets of spike mutations analyzed by PSQ. PSQ generated interpretable pyrograms and enabled reliable detection of predefined VOC-associated spike mutations. Direct comparison of PSQ calls with the corresponding WGS-derived sequences showed 98.6% concordance (489/496 evaluable comparisons). The workflow enabled same-day mutation profiling directly from diagnostic material, providing rapid sequence-level information on mutations associated with increased transmissibility and immune escape. These findings demonstrate that PSQ provides rapid and reliable screening of predefined spike mutations associated with early SARS-CoV-2 VOCs. The workflow may complement whole-genome sequencing in surveillance settings where rapid mutation-level information is required. In a broader sense, the study demonstrates a data-driven approach for developing and periodically adapting targeted molecular surveillance assays as viral populations evolve.