人肺炎衣原体LAMP快速检测方法的建立

Establishment of a loop- mediated isothermal amplification assay for rapid detection of Chlamydia pneumoniae

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 loop-mediated isothermal amplification assay for rapid detection of Chlamydia pneumoniae. Methods According to the Ompl gene sequences of Chlamydia pneumoniae available in GenBank, primers specific to the conservative area of Ompl gene were designed using PrimerExplorer V4 soft, the detection mastermix and reaction conditions were optimized, and the specificity and sensitivity were verified. Results Chlamydia pneumoniae was detected and there was no cross-reaction with Influenza virus, Parainfluenza virus, Respiratory syneytial virus, Adenoviruses and Human bocavirus. The detection limit of LAMP assay was 50 copies/μL. On the other hand, the amplification could be finished within 50 min and with the addition of calcein, the presence of Chlamydia pneumoniae could be detected by naked eyes. Conclusions The newly developed LAMP assay is a specific, sensitive, rapid and simple method for detection of Chlamydia pneumoniae in field conditions.