Mycoplasma pneumoniae (M. pneumoniae), a major human respiratory pathogen, necessitates the development of rapid point-of-care testing (POCT) platforms for clinical management. However, current two-step workflows suffer from operational complexity and aerosol contamination risks. This limitation stems from CRISPR-Cas12 mediated template degradation in single-reaction systems, which compromises amplification efficiency and detection sensitivity. Here, we combined RPA and CRISPR Cas12b by leveraging the difference in their optimal temperatures to construct a novel TRACER (Thermo-activated RPA Amplification for CRISPR-Cas12b Efficient Recognition) technology. Through precise temperature modulation, TRACER sequentially executes isothermal amplification and CRISPR-mediated detection while preventing premature template cleavage, thereby maintaining optimal reaction efficiency. The platform demonstrates exceptional analytical sensitivity with a detection limit of 1 copy/μL, representing a 100-fold improvement over conventional one-pot RPA-CRISPR-Cas12a systems. Clinical validation using 195 specimens revealed diagnostic performance metrics of 99.2 % sensitivity (119/120), 100.0 % specificity (75/75), and 99.5 % accuracy (194/195). This innovative combination of single-tube reaction, field-deployable instrumentation, and cost-effectiveness establishes TRACER as an ideal POCT solution for M. pneumoniae detection in diverse clinical settings.
