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目的采用环介导等温扩增(LAMP)技术建立针对人肺炎衣原体基因的特异、灵敏、快速、可视的检测方法。方法对Gen Bank登录的肺炎衣原体的Omp1基因序列进行生物学分析,利用Primer Explorer V4软件,针对保守区域设计LAMP引物,建立并优化反应条件,对其特异性、灵敏度和稳定性进行评估。结果该方法能特异性扩增出肺炎衣原体,而对流感病毒、副流感病毒、呼吸道合胞病毒、人腺病毒、人博卡病毒均无特异反应,其最低检测限度为50copies/μL。另外,反应周期可在50 min内完成,并通过反应液中加入荧光染料来对结果进行可视化判读。结论本研究建立的针对人肺炎衣原体的LAMP检测方法具有特异性强、灵敏度高、简便快速、可视化判读结果、成本低等优势,为基层或现场进行肺炎衣原体的快速确诊提供了可靠的技术支撑。
Objective To establish a specific, sensitive, rapid and visual method for detection of Chlamydia pneumoniae gene using ring-mediated isothermal amplification (LAMP) technique. Methods The gene sequence of Omp1 gene of Chlamydia pneumoniae was detected by Gen Bank. Primer Explorer V4 software was used to design LAMP primer for conserved region. The reaction conditions were established and optimized to evaluate its specificity, sensitivity and stability. Results The method was able to amplify Chlamydia pneumoniae specifically and showed no specific response to influenza virus, parainfluenza virus, respiratory syncytial virus, human adenovirus and human Boka virus. The limit of detection was 50copies / μL. In addition, the reaction cycle can be completed within 50 min, and visualization of the results by adding a fluorescent dye to the reaction solution. Conclusion The LAMP detection method for Chlamydia pneumoniae established in this study has the advantages of high specificity, high sensitivity, simple and rapid, visual interpretation results and low cost. It provides a reliable technical support for the rapid diagnosis of Chlamydia pneumoniae at the grassroots level or in the field.