Endpoint genotyping of A2063G and A2064G mutations in Mycoplasma pneumoniae

Macrolide resistance in Mycoplasma pneumoniae is primarily associated with point mutations in domain V of the 23S rRNA gene, most commonly A2063G and A2064G. Rapid detection of these mutations is important for appropriate antimicrobial therapy. In this study, we developed a fluorescence-based endpoint genotyping assay using minor groove binding (MGB) probe–based PCR to detect mutations at positions 2063 and 2064 in the M. pneumoniae 23S rRNA gene. We designed position-specific MGB probes to selectively bind mutant sequences at positions 2063 and 2064, allowing for the accurate discrimination of point mutations at adjacent nucleotide sites. To achieve clear genotype-specific fluorescence separation, we optimized the assay conditions. The detection limits were 31.0, 27.2, 43.2, 39.3, and 32.7 copies/mL for the wild-type, A2063G, A2063C, A2063T, and A2064G genotypes, respectively. We observed no cross-reactivity among 24 non-target respiratory pathogens. Clinical evaluation was performed using 44 respiratory specimens obtained from 44 patients with suspected M. pneumoniae infection. Sanger sequencing yielded interpretable results for 40 specimens, and the developed assay was fully concordant with the sequence-based reference results in all 40. Across all 44 specimens, 100% overall percent agreement (44/44, 95% CI, 92.0–100.0%; Cohen’s κ = 1.00) was additionally observed against a commercially available real-time PCR assay. In summary, this multiplex MGB probe–based PCR assay allows for rapid and reliable detection of closely spaced macrolide resistance mutations in M. pneumoniae and could support timely antimicrobial decision-making in clinical settings. Key points Minor groove binder polymerase chain reaction detects macrolide resistance.Decreasing primer concentrations improved probe–target discrimination.Genotype assignment can be performed using endpoint fluorescence without melting-curve analysis.