Enhanced SfaTnpB enables single-base-specific, one-pot nucleic acid detection for high-sensitivity diagnostics.

CRISPR/Cas12-based nucleic acid detection has revolutionized molecular diagnostics but shows limited single-nucleotide specificity, limited high-fidelity subtype discrimination, and limited compatibility with one-pot assays, restricting its broader clinical application. Here, we report a transposon-associated transposase B (TnpB) ortholog, SfaTnpB, with high trans-cleavage activity, robust single-base mismatch discrimination, and broad temperature tolerance. By stepwise engineering of its guide RNA (ωRNA), we developed an enhanced SfaTnpB (enSfaTnpB) system with markedly improved trans-cleavage efficiency. In combination with a TAM-independent split-activator strategy, this system enables precise detection of single-nucleotide polymorphisms. We further developed TOPIC (TnpB-based One-Pot nucleIC acid detection), a one-pot detection platform coupling enSfaTnpB with recombinase-aided amplification (RAA) or loop-mediated isothermal amplification that enables ultrasensitive detection of human papillomavirus (HPV) subtypes 16 and 18 (∼4 copies/μl) and African swine fever virus DNA (∼3 copies/μl). Finally, RAA-TOPIC accurately detected and genotyped 14 high-risk HPV subtypes with high-fidelity subtype discrimination, showing complete concordance with quantitative real-time PCR-based clinical diagnostics. These findings establish TOPIC as a compact, programmable, and scalable molecular detection tool with broad potential for precision diagnostics and point-of-care testing, particularly in resource-limited settings.