A novel thermo-activated one-pot RPA-CRISPR-Cas12b assay for Mycoplasma pneumoniae POCT

Aug 8, 2025Biosensors & bioelectronics

A new heat-activated one-step test using CRISPR for quick detection of Mycoplasma pneumoniae

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Abstract

The TRACER technology achieves a detection limit of 1 copy/μL, significantly enhancing sensitivity for Mycoplasma pneumoniae detection.

TRACER combines isothermal amplification and CRISPR detection to improve reaction efficiency.
This method prevents premature template degradation, which is a limitation in existing systems.
Clinical validation with 195 specimens showed 99.2% sensitivity and 100.0% specificity.
The platform represents a 100-fold improvement over conventional one-pot RPA-CRISPR-Cas12a systems.
TRACER offers a single-tube reaction approach that is suitable for point-of-care testing.

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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.

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A novel thermo-activated one-pot RPA-CRISPR-Cas12b assay for Mycoplasma pneumoniae POCT

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