Rapid, ultrasensitive and highly specific diagnosis of Mycoplasma pneumoniae by a CRISPR-based detection platform

Area of Science:

• Molecular Biology
• Infectious Diseases
• Diagnostics

Background:

• Mycoplasma pneumoniae (MP) causes significant morbidity and mortality, particularly in vulnerable age groups.
• Accurate, sensitive, and rapid diagnostic methods for MP infection are currently lacking.
• Existing diagnostic tools may not be suitable for all clinical or field settings.

Purpose of the Study:

• To develop a novel CRISPR-Cas12b-based detection platform integrated with recombinase polymerase amplification (RPA) for diagnosing MP infection.
• To establish a rapid, simple, and highly sensitive diagnostic assay for MP.
• To evaluate the clinical performance of the developed assay using patient samples.

Main Methods:

• Developed the MP-RPA-CRISPR assay combining RPA for amplifying the MP CARDS toxin gene and CRISPR-Cas12b for detection.
• Optimized RPA reaction conditions, including temperature (37°C).
• Validated the assay's sensitivity (down to 5 fg MP genomic DNA), specificity, and performance on 96 bronchoalveolar lavage fluid (BALF) samples.

Main Results:

• The MP-RPA-CRISPR assay demonstrated high sensitivity, detecting as little as 5 fg of MP genomic DNA.
• The assay accurately distinguished MP strains from non-MP strains without cross-reactivity.
• Clinical evaluation on 96 BALF samples showed perfect concordance with microfluidic chip technology, correctly identifying 45 MP-infected and 51 non-MP infected cases.

Conclusions:

• The MP-RPA-CRISPR assay is a simple, rapid, portable, and highly sensitive method for MP infection diagnosis.
• This assay shows significant potential for use in diverse settings, including clinical laboratories, field applications, and resource-limited areas.
• The developed platform offers a promising advancement in the timely and accurate diagnosis of Mycoplasma pneumoniae infections.