MCDA-CRISPR-Cas12-based diagnostic platform for detection of Mycoplasma Pneumoniae Targeting the CARDS gene

Abstract Mycoplasma pneumoniae (MP) is a leading cause of pediatric community-acquired pneumonia (CAP) and advanced techniques for Mycoplasma pneumoniae pneumonia (MPP) diagnosis are urgently needed. Here, a novel diagnostic test combined multiple cross displacement amplification ( M CDA) with C RISPR-Cas12a system targeting the C ARDS gene of MP , termed MP-MCC, were developed for MPP detection. The MCDA assay was employed for nucleic acid amplification, and the CRISPR-Cas12a/CrRNA complex was used to decode the amplification products. Then, the detection result was observed via real-time fluorescence. The optimal conditions for our assay include a MCDA reaction at 61°C for 40 min and a CRISPR detection at 37°C for ~ 5 min. The results showed that reaction products were detectable from as little as 10fg of pure MP templates and from approximately 10 copies of plasmids containing CARDS genes. The specificity in detecting MP strains was 100%, and there was no cross-reaction to non-MP strains. Furthermore, our assay was further validated using clinical samples, which offered high sensitivity and specificity for MPP diagnostic. Taken together, the detection technique developed here has advantages on rapidity, sensitivity and specificity for MP detection, which could be used as a potential tool for clinical MPP diagnosis.