A CRISPR-Cas12a—Based platform for ultrasensitive, rapid, and highly specific detection of Mycoplasma pneumonia in clinical application
Nan Jia, Juan Zhou, Fei Xiao, Baoying Zheng, Xiaolan Huang, Chunrong Sun, Jin Fu, Xu Zheng, Min Chen, Yi Wang
Abstract
Mycoplasma pneumoniae (MP), which is responsible for a majority of community-acquired pneumonia (CAP) in children, has been largely underestimated. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform termed MP-MCDA-CRISPR assay for MP infection diagnosis and clinical application. The MP-MCDA-CRISPR assay amplified the CARDS gene of MP by MCDA method, followed by trans -cleavage of the reporter molecular upon the formation of CRISPR-Cas12a-gRNA-target DNA complex, which was confirmed by the release of fluorescent signals. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the CARDS gene of MP. The optimal temperature for MCDA pre-amplification is 64°C, and the time for CRISPR-Cas12a-gRNA biosensing process is 5 min. The limit of detection (LoD) of the MP-MCDA-CRISPR assay is 50 fg per reaction without any cross-reaction with other non-MP pathogens. The MP-MCDA-CRISPR assay accurately identified the 50 real time-PCR positive clinical samples and 78 negative ones. Taken together, the MP-MCDA-CRISPR assay designed here is a promising diagnostic tool for point-of care (POC) testing of MP infection.