Rapid, sensitive, and highly specific detection of <i>Mycoplasma pneumoniae</i> and its mutation using the RPA-CRISPR/Cas13a system

(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.