重组酶聚合酶等温扩增快速检测肺炎支原体方法的建立和初步应用

摘要 目的：建立基于重组酶聚合酶扩增技术（RPA）技术快速检测肺炎支原体的方法。方法：本研究以肺炎支原体编码P1黏附蛋白为靶基因，利用Primer Premier 5软件进行引物、探针的设计，最终筛选出最佳引物。同时设计相应的实时荧光定量PCR（RT-PCR）引物用于后续的验证试验。对反应体系试剂比例、反应时间、反应温度、引物探针浓度进行确定。肺炎支原体、解脲支原体、人型支原体、肺炎克雷伯菌、肺炎双球菌、大肠杆菌和链球菌作为对照评估RPA检测肺炎支原体的特异性及敏感度。结果：RPA快速检测肺炎支原体方法仅需14 min，检测灵敏度达200 copies/mL；6种非肺炎支原体均不能扩增，特异性较高。结论：本研究建立了肺炎支原体的RPA快速检测方法，具有迅速、简便、经济等优势，为肺炎支原体的快速检测提供一个新的有利工具。

Establishment and Preliminary Application of Rapid Detection of Mycoplasma Pneumoniae by Recombinase Polymerase Amplification

ABSTRACT Objective: To establish a rapid detection method of mycoplasma pneumoniae based on recombinase polymerase amplification (RPA). Methods: In this study, the P1 adhesion protein encoded by mycoplasma pneumoniae was used as the target gene. Primer premier 5 software was used to design primers and probes, and finally the best primers were selected. At the same time, the corresponding real-time fluorescent quantitative PCR (RT-PCR) primers were designed for subsequent validation tests. The ratio of reagent, reaction time, reaction temperature and the concentration of primer probe were determined. mycoplasma pneumoniae, ureaplasma urealyticum, mycoplasma hominis, klebsiella pneumoniae, diplococcus pneumoniae, escherichia coli and streptococcus were used as controls to evaluate the specificity and sensitivity of RPA in detecting mycoplasma pneumoniae. Results: RPA rapid detection of mycoplasma pneumoniae only took 14 minutes, the detection sensitivity was 200 copies/mL; 6 kinds of non mycoplasma pneumoniae could not be amplified, and the specificity was high. Conclusion: The RPA rapid detection method of mycoplasma pneumoniae was established in this study, which has the advantages of rapidity, simplicity and economy, and provides a new tool for rapid detection of mycoplasma pneumoniae.