Development of real-time fluorescence quantitative PCR for the detection of Mycoplasma pneumoniae

doi:10.3969/j.issn.1007-8134.2016.01.010

Abstract：Objective To develop a new real-time fluorescence quantitative PCR assay for the detection of Mycoplasma pneumoniae, and to compare its detection performance with the kit and traditional nested PCR method. Methods According to the sequence of community-acquired respiratory distress syndrome toxin gene in Mycoplasma pneumoniae, specific primers were designed and the standard plasmids were constructed. The standard curve was drawn in the real-time PCR using SYBR green as fluorescence reporter. And then the sensitivity and specificity of the newly-developed real-time PCR assay were compared with those of the kit and traditional nested PCR method by detecting other bacteria and 170 throat swab samples from child patients. Results In the sensitivity tests, the newly-developed real-time fluorescence PCR and nested PCR could detect 10 copies of FH DNA, while the kit could detect 100 copies. In the specificity tests, no amplification of other Mycoplasma or bacteria was observed using newly-developed real-time fluores cence PCR and the kit. In detecting the 170 throat swab samples from child patients, the positive rates of newly-developed real-time fluorescence PCR, the kit and nested PCR were 60.00%, 50.00% and 53.53%, respectively. The newly-developed realtime fluorescence PCR had good correlation with the kit (r=0.953), and the total consistency rate of these two methods was 86% with Kappa value of 0.729, while that of the newly-developed real-time fluorescence PCR and nested PCR was 90% with Kappa value of 0.797. Conclusions The newly-developed real-time fluorescence PCR assay is more sensitive and cheaper than the kit with the same specificity, and more rapid and laborsaving than the nested PCR in detecting Mycoplasma pneumoniae from the clinical samples. So the newly-developed real-time fluorescence PCR assay can be used widely in scientific research and clinical application.