A multisite SNP genotyping and macrolide susceptibility gene method for Mycoplasma pneumoniae based on MALDI-TOF MS

Abstract

In this study, a multisite SNP genotyping and macrolide (ML) susceptibility gene test method for Mycoplasma pneumoniae (M. pneumoniae) was developed based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The detection limit of this method for nucleic acids was 10² -10³ copies/reaction. Six SNP site-based genotyping and 3 ML susceptibility sites could be detected simultaneously based on multiplex PCR and mass probe. Using the method constructed in this study, 141 Chinese clinical isolates were divided into 8 SNP types. All the SNP test results for the ML susceptibility gene were in line with those of the 23S rRNA sequencing results. With this method, the multisite SNP genotyping and ML susceptibility determination of M. pneumoniae can be completed simultaneously in one test, which greatly reduces the workload and cost, improves the genotyping ability of M. pneumoniae and deserves clinical application.

Keywords: Biotechnology; Microbial Genetics; Microbiology.

Graphical abstract

Figure 1

MS peak of the MPE probes (A) MS peaks of 9 MPE probes without extension; (B) SNP peak of the 9 MPE probes extended with M. pneumoniae M129.

Figure 2

Detection of 9 SNP sites in the presence of different nucleic acid from M. pneumoniae based on the PCR-MALDI-TOF MS method The test was performed twice for each concentration. a-g: of a concentration range of 10⁵-10⁰copies/μL and the blank control.