Rapid, sensitive, and highly specific detection of Mycoplasma pneumoniae and its mutation using the RPA-CRISPR/Cas13a...

Mycoplasma pneumoniae (MP) is an important pathogen associated with respiratory tract infections. Macrolide-resistant MP infections (which mainly contain the A2063G mutation in their 23S rRNA) contribute to severe cases, complications, and sequelae development. Therefore, the development of a sensitive, rapid, and accurate method for MP detection and its A2063G mutation is critical for early differential diagnosis and for guiding clinical medication. Herein, we establish a one-step, highly efficient, and specific fluorescence-based detection system for the MP nucleic acid using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a combined with recombinase polymerase amplification (RPA) isothermal technology. A detection limit of 5 copies/μL within 40 min was achieved using this system. When applied to 57 clinical specimens, this method exhibited a sensitivity of 96.3% and a specificity of 100% compared with quantitative real-time polymerase chain reaction (PCR). We also developed a two-step detection system for macrolide-resistant mutations with a sensitivity of 10 copies/μL. The RPA-CRISPR/Cas13a system was further applied to detect 27 clinical specimens, and the results were consistent with the sequencing data. Additionally, the RPA-CRISPR/Cas13a detection system was integrated with lateral flow assay (LFA) technology to develop an LFA-RPA-CRISPR/Cas13a detection system. In conclusion, our RPA-CRISPR/Cas13a detection system enabled rapid, sensitive, and highly efficient detection of MP and its mutation. This new method provides an efficient tool for clinical and point-of-care testing (POCT) to facilitate early treatment and infection control.IMPORTANCEA rapid, sensitive, and highly efficient nucleic acid detection system for Mycoplasma pneumoniae (MP) was developed in this study by integrating clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a with recombinase polymerase amplification (RPA) technology. A two-step assay based on this technology was then further developed to identify the A2063G mutation in the 23S rRNA gene of MP that was associated with macrolide resistance. The proposed system was then validated using clinical samples, and it demonstrated consistency with conventional diagnostic and sequencing results. Therefore, the RPA-CRISPR/Cas13a system enabled rapid, sensitive, and cost-effective detection of MP and its drug resistance mutation. This method is a robust tool for clinical and point-of-care testing (POCT) applications. These systems will facilitate early treatment and infection control of MP.