中华皮肤科杂志 ›› 1997, Vol. 30 ›› Issue (3): 160-162.

• 论著 • 上一篇    下一篇

竞争性聚合酶链反应定量检测沙眼衣原体

李珊山, 叶顺章   

  1. 中国医学科学院、中国协和医科大学皮肤病研究所, 南京210042
  • 收稿日期:1996-07-11 修回日期:1996-12-03 出版日期:1997-06-15 发布日期:1997-06-15

Quantitative Detection of Chlamydia Trachomatis by Competitive Polymerase Chain Reaction

Li Shanshan, Ye Shunzhang   

  1. Institute of Dermatology, Chinese Academy of Medical Sciences, Peking Union Medical College, Nanjing 210042
  • Received:1996-07-11 Revised:1996-12-03 Online:1997-06-15 Published:1997-06-15

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摘要： 为了便于研究沙眼衣原体(CT)感染的致病机理,我们建立了一种竞争性聚合酶链反应(PCR)定量检测CT的方法。合成、克隆并定量了一与CT主要外膜蛋白基因(ompl)靶序列具有相同引物位点的内参标(IS),通过共同扩增已知数量克隆的CTompl野生型模板和IS,构建竞争性PCR标准曲线。恒定拷贝数(1000个分子)的IS与每一待测模板共同扩增,用靶序列与IS的PCR产物的密度比值定量标本中的CT.对11份培养阳性的泌尿生殖道标本进行了CT的定量检测。结果发现,一个培养包涵体形成单位大约相当于75个DNA分子。研究表明:竞争性PCR提供了一种可靠且敏感、可定量临床标本中CT的方法。

关键词: 沙眼衣原体, 外膜蛋白基因, 聚合酶链反应

Abstract: In order to facilitate analysis of pathogenesis of chlamydial infection, a competitive polymerase chain reaction for the quantitative detection of C.trachomatis was developed. An internal standard(IS) that has the same primer template as the target of the major outer membrance protein gene(ompl) of C.trachomatis was synthesized, cloned and quantified. A standard curve was constructed by co-amplification of known quantities of the cloned ompl target template with IS. Absolute quantitation of the test sample was achieved by co-amplification of constant number of IS with the wild-type target and determining the wild-type target/IS ratio of densitometric value. The number of inclusions in culture and DNA copies in PCR was measured in 11 urogenital specimens. The results show that the DNA copy number per inclusion was approximately 75. This study indicates that competitive PCR provides a reliable and sensitive method of quantitating the number of C.trachomatis in clinical specimens.

Key words: Chlamydia trachomatis, Outer membrane protein gene, Polymerase chain reaction