利用SYBR Green检测衣原体Real-Time PCR方法的建立

Development of a Real-Time PCR Assay for Detection and Quantitation of Chlamydia Using SYBR Green and the Light Cycler

利用SYBR Green建立了检测种特异性衣原体的Real-Time PCR方法。本方法应用衣原体种特异性的高度保守特异引物，能够扩增627bp特异片段；使用定量标准基因组DNA，本方法能准确检测最少250fg衣原体DNA。Real-TimePCR方法与免疫荧光方法的检测结果表明：检测4种衣原体临床样本，Real-TimePCR敏感性均在96％～98%；特异性均为100％；这两种方法符合率达97％以上（n=60）；批内和批间重复性试验结果表明，本方法具有良好的准确性。本方法的建立对于快速、准确检测临床样本种特异性衣原体提供了一种切实有效的方法。

Here, we presented a Chlamydiaceae-specific 23S rRNA--based real-time PCR assay for simultaneous detection and quantltation of four members of Chlamydiaceae family, C. trachomatis, C. psittaci, C. pneumoniae and C. Decorum, using SYBR Green and LightcyclerTM. The as- say was characterized using plasmid constructs of the bacteria and verified on standard strains of all four species of the Chlamydiaceae and a large cohort of clinical samples collected from human and animals by comparison with fluorescence immunohistochemistry method. The results showed that the present real-time PCR assay was of high specificity and sensitivity. It was capable of detecting as few as 250 fg of chlamydial DNA （equivalent to 10^-1 IFU） and was applicable to both liquid cultures and clinical samples. This assay may therefore offer a rapid, economic and reliable method for screening of the chlamydiaceae pathogens.