Development and clinical application of real-time quantitative PCR for the detection of mycoplasma pneumonia

Abstract

Objective To develope a new Real-time quantitative PCR assay using SYBR green as fluorescence reporter, which is rapid, specific, sensitive, cheap and accurate for the detection of mycoplasma pneumoniae(MP), and evaluated its clinical application value. Methods The sequence of the 23S rRNA gene in MP type strain FH was selected as amplified regions, and specific primers were designed. Then the related plasmids were extracted as standards, and the absolute quantitative standard curve was established. The sensitivity, specificity of the fluorescence quantitative PCR assay was compared with the nest-PCR and kit; To calculate correlation coefficient, coincidence rate and kappa coefficient, clinical samples were detected using above-mentioned methods and cultivation, respectively. Results The detection sensitivity of the new real-time PCR and nest-PCR was 10 copies of FH DNA, while the kit 100 copies. In the specificity tests, the MP sample was positive, while mycoplasma hominis and other four bacteria were all negative. We applied this real-time PCR assay, nest-PCR, kit and cultivation to 182 clinical specimens, and the detection rates were 55.49%, 52.75%, 47.25% and 39.01%, respectively. The total consistency rate and Kappa coefficient of the new real-time PCR method and nest-PCR were 89.6%, 0.790, respectively; while those of the new method and cultivation were 83.5%, 0.678, respectively. The total consistency rate and Kappa coefficient of the new real-time PCR method and the kit were 89.6%, 0.792, respectively; and the correlation coefficient of these two methods was 0.923, P<0.001. Conclusion Compared with other methods, the new real-time PCR assay could be used to detect mycoplasma pneumoniae quickly and economically, with high sensitivity and specificity, revealing great utility value on varied instrumentation platforms. Key words: Mycoplasma pneumoniae; Children; Real-time PCR; Diagnosis