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Updated: Jun 15, 2025
Optimized PCR-based Detection of Mycoplasma
Published on: June 20, 2011
An accurate and convenient method for Mycoplasma pneumoniae via one-step LAMP-CRISPR/Cas12b detection platform
Tao Liu1, Qing Liu2, Fuqun Chen1
1State Key Laboratory of Pathogenesis, Prevention, Treatment of High Incidence Diseases in Central Asian, Department of Clinical Laboratory, First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang Uygur Autonomous Region, China.
Introduction:
Mycoplasma pneumoniae (MP) is the major cause of respiratory infections that threaten the health of children and adolescents worldwide. Therefore, an early, simple, and accurate detection approach for MP is critical to prevent outbreaks of MP-induced community-acquired pneumonia.
Methods:
Here, we explored a simple and accurate method for MP identification that combines loop-mediated isothermal amplification (LAMP) with the CRISPR/Cas12b assay in a one-pot reaction.
Results:
In the current study, the whole reaction was completed within 1 h at a constant temperature of 57°C. The limit of detection of this assay was 33.7 copies per reaction. The specificity of the LAMP-CRISPR/Cas12b method was 100%, without any cross-reactivity with other pathogens. Overall, 272 clinical samples were used to evaluate the clinical performance of LAMP-CRISPR/Cas12b. Compared with the gold standard results from real-time PCR, the present method provided a sensitivity of 88.11% (126/143), specificity of 100% (129/129), and consistency of 93.75% (255/272).
Discussion:
Taken together, our preliminary results illustrate that the LAMP-CRISPR/Cas12b method is a simple and reliable tool for MP diagnosis that can be performed in resource-limited regions.
Insights
A new diagnostic method combines loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12b for rapid and accurate detection of Mycoplasma pneumoniae (MP). This one-pot assay offers high sensitivity and specificity, making it ideal for resource-limited settings.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Mycoplasma pneumoniae (MP) is a significant cause of respiratory infections in children and adolescents globally.
- Early and accurate MP detection is crucial for managing community-acquired pneumonia and preventing outbreaks.
Purpose of the Study:
- To develop a simple, rapid, and accurate diagnostic method for Mycoplasma pneumoniae.
- To combine loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12b assay in a one-pot reaction for MP identification.
Main Methods:
- A one-pot reaction integrating LAMP and CRISPR/Cas12b was developed for MP detection.
- The assay was optimized to run at a constant temperature of 57°C within 1 hour.
- Clinical performance was evaluated using 272 samples and compared against real-time PCR.
Main Results:
- The LAMP-CRISPR/Cas12b assay demonstrated a limit of detection of 33.7 copies per reaction.
- 100% specificity was observed, with no cross-reactivity with other common pathogens.
- Clinical evaluation showed 88.11% sensitivity, 100% specificity, and 93.75% consistency compared to real-time PCR.
Conclusions:
- The LAMP-CRISPR/Cas12b method is a simple, reliable, and accurate tool for Mycoplasma pneumoniae diagnosis.
- This assay is suitable for use in resource-limited settings, aiding in early detection and outbreak prevention.
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