实时荧光定量PCR检测沙眼衣原体的研究

目的采用TaqMan探针建立检测沙眼衣原体的实时荧光定量PCR（real-time PCR）方法。方法根据沙眼衣原体外膜蛋白A的基因（ompA）序列设计引物和探针,以克隆的ompA部分基因片段作DNA模板,建立实时荧光定量检测方法。结果建立的荧光定量PCR检测方法的最低检出限为5 copies/反应,检测线性范围100～107线性关系良好（r2=0.997）,比巢式PCR敏感100倍;且与鹦鹉热衣原体、淋球菌、解脲脲原体、大肠杆菌等病原菌DNA以及人基因组DNA均无交叉反应,表明该方法具有良好的特异性。以巢式PCR作参比,建立的荧光定量PCR法检测沙眼衣原体的阳性符合率为100.00%,阴性符合率为95.09%,总符合率为96.81%。结论建立的检测沙眼衣原体实时荧光定量PCR具有特异性强和敏感性高的特点,可快速检测样本中微量沙眼衣原体DNA,适用于对沙眼衣原体进行大规模筛选。

Detection of Chlamydia trachomatis by real-time PCR

Objective In this study, we developed a real-time polymerase chain reaction （real-time PCR） based method for the detection of Chlamydia trachomatis. Methods The primers and TaqMan probes were designed based on ompA gene of Chlamydia trachomatis. Results For the quantitative real-time PCR, the relationship between the values of threshold cycle （Ct） and the DNA copy number was linear （r2=0.997） and the sensitivity was at 5 copies / reaction. The sensitivity was about 100 times higher than the nested PCR. The real-time PCR reacted negatively to the genomic DNA of Chlamydia psittaci, Neisseria gonorrhoeae, Ureaplasma urealyticum, bacterial agents and human. Compared with nested PCR, the positive rate for the detection of clinical specimens was 100.00%, and the negative coincidence rate was 95.09%. Conclusion The developed real-time PCR is highly specific and sensitive for the detection of Chlamydia trachomatis. It can be used for the detection of Chlamydia trachomatis in cervical secretions from patients with suspected Chlamydia trachomatis infection.