Palm-sized CRISPR sensing platform for on-site Mycoplasma pneumoniae detection

Pneumonia remains a prevalent childhood illness and is the foremost cause of mortality due to infectious diseases among children under the age of five. Mycoplasma pneumoniae (M. pneumoniae) causes the most frequent type of atypical pneumonia in this age group, has raised global health concern. Therefore, there is a pressing need for a rapid, low cost, and user-friendly method for the early diagnosis of M. pneumoniae pneumonia. Herein, we develop a CRISPR sensing platform for on-site M. pneumoniae detection, termed CRAFT (Crispr-based rapid assay device for field testing). The CRAFT provides "sample in-result out" functionality. It completed sample processing and nucleic acid extraction within 5 min at room temperature, with efficiency comparable to commercial kits. RPA and CRISPR/Cas12a reagents were isolated in a closed tube using a movable magnetic bead valve, and the RPA product was then mixed with the CRISPR reagent. The limit of detection for M. pneumoniae using CRAFT was 100 copies/μL, and the method exhibited no cross-reactivity with other respiratory pathogens. CRAFT was utilized to validate 50 clinical samples, and the results demonstrated 100 % consistency with those obtained by qPCR. This versatile platform holds significant potential for point-of-care testing of M. pneumoniae, particularly in resource-limited settings.