肺炎衣原体实时定量PCR检测方法的建立

目的:探讨实时定量PCR检测肺炎衣原体(chlamydia pneumonia,Cpn)的方法.方法:根据GenBank提供的肺炎衣原体基因序列,选取高特异性和保守型的区域进行引物设计,并建立实时定量PCR(real-time PCR,RT-PCR)的检测方法.同时用肺炎支原体、肺炎双球菌、流感病毒、副流感病毒以及腺病毒对Cpn引物的特异性进行分析.对呼吸道感染患者200个咽拭子样本和68个肺泡冲洗液样本进行检测,以荧光抗体检测法作对照,评价实时定量PCR检测Cpn的准确性.结果:Cpn PCR引物对肺炎支原体、肺炎双球菌、流感病毒、副流感病毒以及腺病毒均显示阴性,对Cpn显示为阳性,特异性良好;荧光实时定量PCR技术检测的准确性优于荧光抗体检测法.结论:荧光实时定量PCR技术检测Cpn体灵敏度高、周期短,可作为临床诊断Cpn的手段.

Detection of chlamydia pneumonia with real-time PCR

Objective: To investigate real-time PCR as a method to detect chlamydia pneumonia in clinical settings.Methods: Primers were designed according to conserved DNA region of chlamydia pneumonia from GenBank.Mycoplasma pneumonia,pneumococci,influenza B virus,para influenza virus and adenovirus were used to examine the specificity of primers.Two hundred and sixty-eight patients with respiratory tract infections were determined by real-time PCR and fluorescent antibody.Accuracy of measurement was compared between real-time PCR and fluorescent antibody.Results: Real-time PCR showed that primer specificity on Mycoplasma pneumonia,Pneumococci,Influenza virus,Para influenza virus and Adenovirus were negative but was positive on Chlamydia pneumonia.The primers were only specific for Chlamydia pneumonia.The diagnostic accuracy of real-time PCR was better than that of immunofluorescence.Conclusions: Real time quantitative PCR can be used as a clinical diagnostic assay with chlamydia pneumonia for high sensitivity and short period.