Development of a Real-Time Polymerase Chain Reaction Assay to Detect Mycoplasma pneumoniae Using the Panther Fusion System.

Background/Objectives: Mycoplasma pneumoniae is a major cause of community-acquired pneumonia, and the increasing prevalence of macrolide-resistant M. pneumoniae (MRMP) has created a need for rapid assays that simultaneously detect the pathogen and resistance-associated mutations. This study developed and validated a fully automated laboratory-developed test (LDT) using the Hologic Panther Fusion® system. Methods: The automated MP assay targeting domain V of the 23S rRNA gene was implemented on the Panther Fusion® platform. Analytical performance was evaluated by determining the limit of detection (LoD), repeatability, reproducibility, and cross-reactivity. Clinical performance was assessed using 419 nasopharyngeal swab specimens (90 positive and 329 negative) and compared with conventional real-time PCR. Mutation status was confirmed by sequencing. Results: The automated MP assay detected wild-type, A2063G, and A2064G targets with an LoD of approximately 1 × 102 copies/μL and showed no cross-reactivity with 16 respiratory pathogens. Clinical sensitivity and specificity were 96.7% (87/90) and 100% (329/329), respectively, with high concordance with conventional real-time PCR and sequencing. Conclusions: The automated MP assay provides rapid and accurate detection of M. pneumoniae and major macrolide resistance mutations, offering a practical molecular diagnostic solution for routine clinical laboratories.