As the COVID-19 pandemic continues to challenge global health systems, the reliability of diagnostic tests remains a critical concern. The most accurate way to identify SARS-CoV-2 infection is nucleic acid amplification tests (NAATs), especially real-time PCR (RT-PCR) assays. However, changes in SARS-CoV-2 primer and probe binding sites might compromise the accuracy of these diagnostic tests and increase false-negative rates. Real-time PCR serves as the gold standard for SARS-CoV-2 detection but shows 2–29% false-negative rates. The present study analyzed ~ 26,000 SARS-CoV-2 genomic sequences from the Global Initiative on Sharing All Influenza Data (GISAID) database to shed light on genetic variants that affected the performance of ongoing setup RT-PCR primer and probe set. This study assesses 12 primer sets for detecting SARS-CoV-2 variants from late 2019 to early 2023 across four frameworks: chronological, geographical, variant-wise, and diagnostic metrics. We validated computational predictions using clinical specimens and Sanger sequencing. Our findings indicate a correlation between amplification failures and single-point mutations or other genetic alterations in the primer and probe binding sites, leading to false-negative results in RT-PCR testing. Our findings provide crucial data for RT-PCR assay design and enhancement. Specifically, our analysis provided quantitative mismatch rates (0.15–77.15%), identified critical binding site mutations causing RT-PCR failures, and established temporal performance patterns tracking variant-driven primer degradation. These results enable evidence-based primer selection and highlight the need for continuous surveillance in viral pandemics. These findings recommend implementing multiplex RT-PCR assays and continuous primer surveillance for reliable COVID-19 diagnosis.