A Novel Detection Procedure for Mutations in the 23S rRNA Gene of Macrolide-Resistant Mycoplasma pneumoniae with Two Non-Overlapping Probes Amplification Assay

Mycoplasma pneumoniae is a significant cause of community-acquired pneumonia, which is often empirically treated with macrolides (MLs), but, presently, resistance to MLs has been a matter of close clinical concern. This assay is intended to contribute to resistance detection of M. pneumoniae in clinical practice. A novel real-time PCR assay with two non-overlapping probes on the same nucleic acid strand was designed in this study. It could effectively detect all mutation types of M. pneumoniae in 23S rRNA at loci 2063 and 2064. The results were determined by the following methods: Delta CT < 0.5 for MLs-sensitive M. pneumoniae; Delta CT > 2.0 for MLs-resistant M. pneumoniae; 10 copies as a limit of detection for all types. For detection of M. pneumoniae in 92 clinical specimens, the consistency between the results of this assay and the frequently used real-time PCR results was 95.65%. The consistency of MLs resistance results between PCR sequencing and this assay was 100% in all 43 specimens. The assay could not only cover a comprehensive range of targets and have high detection sensitivity but is also directly used for detection and MLs analysis of M. pneumoniae in specimens.