A method for rapid detection of Mycoplasma pneumoniae and its macrolide resistance mutation / 中华检验医学杂志

ABSTRACT

Objective To develop a method for rapid detection of Mycoplasma pneumoniae and its macrolide resistance mutation. Methods The primers and cycling probe sets were designed to detect two single nucleotide mutation, A2063G and A2064G, in the 23s rRNA gene of Mycoplasma pneumoniae. By using recombinant plasmids containing 23s rRNA gene fragments, 102 Mycoplasma pneumoniae clinical isolates from 2005 to 2008, and 136 nasopharyngeal suction specimens from pediatric patients with low respiratory tract infections in Shanghai Children's Hospital from November to December in 2009 were investigated to determine the specificity and the sensitivity of the CycleavePCR method. PCR amplification and sequence analysis of 23S rRNA genes were performed for all Mycoplasma pneumoniae strains and Mycoplasma pneumoniae positive specimens to confirm the results of the CycleavePCR method. Results Of 102 clinical isolates, 83 was resistant to erythromycin and sequence results show that all macrolide-resistant Mycoplasma pneumoniae strains harbored an A2063G ( 82/83 ) or A2064G ( 1/83 ) transition mutation in 23S rRNA genes. Twelve was Mycoplasma pneumoniae detected positive by CycleavePCR in 136nasopharyngeal suction specimens. The CycleavePCR results were consistent with those of routine PCR and sequencing. There was no signal production from other bacterial species. Sensitivity and specificity were 100%. The detection limit of the CycleavePCR was 10 plasmid copies per reaction. Experiment can be done within 1.5 h. Conclusion A novel method is developed to detect erythromycin-resistant strains harboring A2063G and A2064G transition mutation in the 23s rRNA gene using CycleavePCR.