Nucleobase mismatches can jeopardize DNA polymerization specificity, causing mutations and errors in DNA replication and detection. Herein we report the first synthesis of novel 2-Se-thymidine triphosphate (SeTTP), describe the single-selenium atom-specific modification strategy (SAM) against T/G mismatches, and demonstrate SAM-assisted polymerization and detection with much higher specificity and sensitivity. SAM can effectively suppress the formation of non-specific products in DNA polymerization and detection. Thus, SAM enhances the specificity of DNA synthesis by approximately 10 000 fold, and in turn, it allows the detection of clinical COVID-19 viral RNA in low copy numbers (single-digit copies), while the conventional RT-qPCR does not.