检测肺炎支原体的实时荧光PCR方法的建立

Detection of Mycoplasma Pneumonia by Fluorescent Real-Time PCR

目的采用Taqman探针技术,组建肺炎支原体荧光PCR基因检测方法。方法对GenBank登录的肺炎支原体的基因序列进行生物信息学分析,针对16S核糖体蛋白基因的保守区域设计引物和探针,组建实时荧光PCR方法检测。采用流感病毒、副流感病毒、呼吸道合胞病毒和腺病毒进行特异性评价。采用倍数稀释的方法进行灵敏度的评价。对来自南方医院的522份临床咽拭子样本进行检测。结果用实时荧光PCR方法检测甲型流感病毒(甲型、乙型)、副流感病毒、呼吸道合胞病毒、呼吸道腺病毒均无特异性反应。该方法可以检测出稀释浓度约为103/mL的肺炎支原体样本。对本院的临床样本检出39份。结论实时荧光PCR检测肺炎支原体具有较高的特异性和灵敏度,可用于肺炎支原体感染的诊断。

Objective To use the Taqman probe technology to detect Mycoplasma pneumoniae by fluorescent real-time PCR（RT-PCR）. Methods The conservative sequence of the 16S rRNA gene of M.pneumoniae was selected to design the specific primers and probe for the detection of M.pneumoniae by real-time RT-PCR. Influenza A virus, influenza B virus, para-influenza virus, respiratory syncytial virus, and adenovirus, were used to evaluate the specificity of the detection method. 522 clinical samples of throat swabs from Guangzhou Nanfang Hospital on 2007 were examined for M.pneumoniae by this detection method. Results The real-time RT-PCR detection of M.pneumoniae was specific and did not cross amplify the influenza A virus, influenza B virus, para-influenza virus, respiratory syncytial virus, and adenovirus. This method can detect down to 10^3/mL of M.pneumoniae. 39 out of 522 cases were positive for M. pneumoniae. Conclusion The real-time RT-PCR with Tagman probe detecting method for M.pneumoniae is specific and sensitive and can be applied for the clinical diagnosis of M.pneumoniae infection.