Molecular beacon based real-time PCR p1 gene genotyping, macrolide resistance mutation detection and clinical characteristics analysis of Mycoplasma pneumoniae infections in children

Abstract

Background: Mycoplasma pneumoniae can be divided into different subtypes on the basis of the sequence differences of adhesive protein P1, but the relationship between different subtypes, macrolide resistance and clinical manifestations are still unclear. In the present study, we established a molecular beacon based real-time polymerase chain reaction (real-time PCR) p1 gene genotyping method, analyzed the macrolide resistance gene mutations and the relationship of clinical characteristics with the genotypes.

Methods: A molecular beacon based real-time PCR p1 gene genotyping method was established, the mutation sites of macrolide resistance genes were analyzed by PCR and sequenced, and the relationship of clinical characteristics with the genotypes was analyzed.

Results: The detection limit was 1-100 copies/reaction. No cross-reactivity was observed in the two subtypes. In total, samples from 100 patients with positive M. pneumoniae detection results in 2019 and 2021 were genotyped using the beacon based real-time PCR method and P1-1 M. pneumoniae accounted for 69.0%. All the patients had the A2063G mutation in the macrolide resistance related 23S rRNA gene. Novel mutations were also found, which were C2622T, C2150A, C2202G and C2443A mutations. The relationship between p1 gene genotyping and the clinical characteristics were not statistically related.

Conclusion: A rapid and easy clinical application molecular beacon based real-time PCR genotyping method targeting the p1 gene was established. A shift from type 1 to type 2 was found and 100.0% macrolide resistance was detected. Our study provided an efficient method for genotyping M. pneumoniae, valuable epidemiological monitoring information and clinical treatment guidance to control high macrolide resistance.

Keywords: Clinical characteristics; Genotyping; Macrolide resistance; Molecular beacon; Mycoplasma pneumoniae.

Fig. 1

Composition of the p1 gene and sequence alignments of primer binding sites. A The illustration of the p1 gene indicating the RepMP4 and RepMP2/3 regions; B Sequence alignments of type 1 and 2 primer and molecular beacon binding sites. Sequence alignment picture was drawn using the tool reported [38]. Blue solid lines were used to indicate binding sequences of type 1 primer and molecular beacon. Green dotted lines were used to indicate binding sequences of type 2 primer and molecular beacon

Fig. 2

Amplification results of gradient positive quality control DNA. A The results of P1-1 amplification; B The standard curve of P1-1 amplification; C The results of P1-2 amplification; D The standard curve of P1- 2 amplification. The numbers 1–10 represent 1 × 10⁹copies/μL, 1 × 10⁸ copies/μL, 1 × 10⁷copies/μL, 1 × 10⁶ copies/μL, 1 × 10⁵copies/μL, 1 × 10⁴ copies/μL, 1 × 10³copies/μL, 1 × 10² copies/μL, 1 × 10¹ copies/μL and 1 copies/μL, respectively. NC was the negative control

Fig. 3

Amplification results of the 100 clinical M. pneumoniae infection samples. A The amplification results of FAM signals with 69 positive samples and 31 negative results; B The amplification results of HEX signals with 31 positive samples and 69 negative results. The figure was drawn using Origin 2016 using the results exported