Development of a multiplex real-time PCR assay for detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and mutations associated with macrolide resistance in Mycoplasma pneumoniae from respiratory clinical specimens

Abstract

The aim of this study was to improve detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in clinical specimens by developing a multiplex real-time PCR assay that includes identification of macrolide-resistant M. pneumoniae. Novel assays targeting a M. pneumoniae conserved hypothetical protein gene, M. pneumoniae 23S rRNA gene mutations associated with macrolide resistance and human β-globin gene (an endogenous internal control) were designed and combined with a previously published C. pneumoniae PCR targeting ompA gene. The resulting quadraplex PCR was validated with a panel of clinical specimens supplemented with external quality assessment specimens, simulated specimens and various bacterial and viral strains. The obtained results were compared to those obtained by reference PCRs or confirmed by sequencing (typing of macrolide resistance). The novel multiplex PCR assay was in 100 % agreement with reference PCRs. Four M. pneumoniae strains with macrolide resistance-associated mutations were identified among 42 strains, which comprises 9.5 % of the study material. Amplification of an internal control excluded sample-derived inhibition possibly leading to false-negative reporting. In conclusion, we have developed a resources conserving multiplex real-time PCR assay for simultaneous detection of M. pneumoniae, C. pneumoniae and the most common mutations leading to macrolide resistance in M. pneumoniae. The assay is a widely useful tool for detection of these respiratory pathogens and will also shed light on the occurrence of macrolide resistance in M. pneumoniae.

Keywords: Chlamydia pneumoniae; Macrolide resistance; Multiplex real-time PCR; Mycoplasma pneumoniae.

Fig. 1

Melting curve displaying a patient sample containing DNA from M. pneumoniae with macrolide resistance-associated mutation (MR–MP). The melting profile of a patient sample was compared to the melting profiles of 3 controls: (1) Plasmid control 2063C which contains M. pneumoniae 23S rRNA PCR target sequence with base C at the position 2063 (DNA amplicon with base C at the position 2064 displays similar melting profile, data not shown). (2) Plasmid control 2063G which contains M. pneumoniae 23S rRNA target sequence with base G at the position 2063 (DNA amplicon with base G at position the 2064 displays similar melting profile, data not shown). (3) Wt (wild type) M. pneumoniae DNA