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Updated: May 13, 2025
Optimized PCR-based Detection of Mycoplasma
Published on: June 20, 2011
Palm-sized CRISPR sensing platform for on-site Mycoplasma pneumoniae detection
Jiajun Li1, Haiqian Huang1, Zerui Song2
1Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200127, China.
Insights
A new CRISPR-based rapid assay device for field testing (CRAFT) enables quick, low-cost detection of Mycoplasma pneumoniae pneumonia in children. This user-friendly platform offers accurate results for early diagnosis, especially in resource-limited settings.
Area of Science:
- Molecular Biology
- Infectious Diseases
- Diagnostics
Background:
- Pneumonia is a leading cause of death in children under five.
- Mycoplasma pneumoniae causes frequent atypical pneumonia, raising global health concerns.
- Current diagnostic methods lack speed, affordability, and user-friendliness for widespread use.
Purpose of the Study:
- To develop a rapid, low-cost, and user-friendly CRISPR sensing platform for on-site Mycoplasma pneumoniae detection.
- To create a "sample in-result out" device for efficient M. pneumoniae diagnosis.
- To address the need for early detection of M. pneumoniae pneumonia, particularly in resource-limited settings.
Main Methods:
- Development of a CRISPR-based rapid assay device for field testing (CRAFT).
- Integration of sample processing, nucleic acid extraction, and CRISPR/Cas12a detection within a single device.
- Utilized recombinase polymerase amplification (RPA) and a magnetic bead valve for reagent isolation and mixing.
Main Results:
- CRAFT achieved sample processing and nucleic acid extraction in 5 minutes at room temperature.
- The limit of detection for M. pneumoniae was 100 copies/μL with no cross-reactivity with other pathogens.
- Clinical validation with 50 samples showed 100% consistency with quantitative PCR (qPCR) results.
Conclusions:
- CRAFT is a highly efficient and accurate platform for the rapid, on-site detection of Mycoplasma pneumoniae.
- The device demonstrates significant potential for point-of-care testing, especially in resource-limited environments.
- This innovation can improve early diagnosis and management of M. pneumoniae pneumonia in children.
Abstract:
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.
