A novel thermo-activated one-pot RPA-CRISPR-Cas12b assay for Mycoplasma pneumoniae POCT
Jiayu Feng, Ze Wu, Wenhui Zhu, Fei‐Fei Jin, Minghai Zhao, Wenjie Zhong, Chen Dai, Yongjian He, Lizhi Yan, Shengquan Wu, Yuhang Wang, Yongyu Rui, Lei Zheng, Qiangqiang Fu
Abstract
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.