Fei Xiao ¹ Yu Zhang ^1* Wenjian Xu ^2* Jin Fu ¹ Xiaolan Huang ¹ Nan Jia ¹ Chunrong Sun ^1* Zheng Xu ^1* Baoying Zheng ¹ Juan Zhou ^1* Yi Wang ^1* Lihui Meng ^2*

• ¹ Capital Institute of Pediatrics, Beijing, China
• ² Children's Hospital of Capital Institute of Pediatrics, Beijing, Beijing Municipality, China

Mycoplasma pneumoniae (M. pneumoniae) is a significant pathogen responsible for community-acquired pneumonia (CAP), predominantly affecting children and adolescents. Here, we devised a rapid method for M. pneumoniae that combined multiple cross displacement amplification (MCDA) with real-time fluorescence technology. A set of ten primers, which was specifically designed for the M. pneumoniae detection, was employed for real-time fluorescence MCDA reaction. Among these, one primer incorporated a restriction endonuclease recognition sequence, a fluorophore, and a quencher, facilitating real-time fluorescence detection. The RT-MCDA reactions were monitored in a simple real-time fluorescence instrument, and conducted under optimized conditions (64 ºC for 40 min). The detection limit of M. pneumoniae RT-MCDA assay for genomic DNA extracted from M. pneumoniae culture is down to 43 fg/µL. This assay accurately identifies M. pneumoniae strains without cross-reacting with other bacteria. To validate its practical application, we tested M. pneumoniae RT-MCDA assay using genomic DNA extracted from clinical samples. The assay's detection capability proved comparable to real-time PCR, MCDAbased biosensor detection, and visual inspection under blue light. The entire process, including rapid DNA extraction and real-time MCDA detection, is completed within an hour. Together, the M. pneumoniae RT-MCDA assay reported here is a simple and effective diagnostic tool for rapid M. pneumoniae detection, which holds significant potential for point-of-care (POC) testing and in resource-limited regions.