A one-pot, visual, multiplex microfluidic digital PCR chip for simultaneous point-of-care detection, genotyping, and macrolide resistance assessment of Mycoplasma pneumoniae

Mycoplasma pneumoniae (M. pneumoniae) is a leading cause of community-acquired pneumonia, especially concerning for young children. Effective management and infection control necessitate rapid, convenient, and user-friendly methods for early diagnosis, including genotype and macrolide resistance monitoring during outbreaks. Current clinical methods are often time-consuming, complex, and lack the capability for simultaneous detection. To address these challenges, we developed a one-pot visual multiplex microfluidic digital polymerase chain reaction (MMDPCR) assay for the simultaneous detection of M. pneumoniae infection, P1 typing, and macrolide resistance, utilizing four special fluorescence probes. The developed multiplex microfluidic digital polymerase chain reaction (MMDPCR) assay provides absolute quantification of clinical samples with a detection time of approximately 1.5 h. The MMDPCR assay demonstrated high specificity and superior sensitivity for M. pneumoniae, with a low detection limit of 10-100 copies and no cross-reactivity with other respiratory pathogens. We validated the assay's performance using 192 clinical samples collected during the 2023-2024 Beijing epidemic. Results showed a positivity rate of 56.25% and a high prevalence (99%) of the A2063 macrolide resistance mutation. Among positive samples, Type I accounted for 75.9% and Type II for 24.1%, showing 100% concordance with qPCR findings. This novel multiplex detection platform holds immense potential for point-of-care testing of M. pneumoniae, offering a rapid and comprehensive tool for analysing its molecular epidemiological characteristics. Its ability to simultaneously provide information on infection, genotype, and macrolide resistance is crucial for guiding clinicians in the rational use of antibiotics and strengthen public health surveillance efforts.