摘要
为建立基于TaqMan MGB探针的沙眼衣原体DNA荧光定量PCR检测方法 ,探讨其临床应用价值 ,用PCR法扩增沙眼衣原体隐蔽质粒pLVG44 0 2 464～ 2 980nt段 ,并克隆入 pMD18 T载体用作参比模板 ,设计一对引物和一个TaqMan MGB探针 ,优化反应条件 ,建立沙眼衣原体DNA荧光定量PCR检测系统 ,并运用该系统同时应用连接酶链式反应 (LCR)法对临床标本进行检测 .结果显示所建立的沙眼衣原体DNA荧光定量PCR检测系统 ,最低检测限度为 1DNA拷贝每反应 ;在 10 0 ～ 10 9DNA拷贝每反应范围内 ,Ct 值 (每个反应管内的荧光信号达到设定的域值时所经历的循环数 )和DNA拷贝数呈线性关系 (r >0 990 ) ;对临床标本检测结果同LCR分析结果吻合率为 10 0 % .以上结果表明 ,所建立的基于TaqMan MGB探针的沙眼衣原体DNA荧光定量PCR检测系统具有敏感性高、特异性强和线性检测范围广等特点 。
To develop a real-time PCR based on TaqMan technology using the new MGB probe for detecting Chlamydia trachomatis DNA, plasmid containing the sequence of interest was constructed for the standardization of the method and to assess its sensitivity. Primers and MGB probe were chosen in the conserved region of cryptic plasmid pLGV440. The Results showed that this Chlamydia trachomatis assay had a threshold sensitivity of one genome copy number per reaction. A linear standard curve was obtained between 10(0) and 10(9) DNA copies/reaction (r > 0.990). Fifty clinical specimens were tested by real-time PCR and LCR simultaneously and the coherence was 100%. These observations suggested that real-time PCR based on MGB probe was an excellent candidate for a standard Chlamydia trachomatis detection method in a large scale.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2003年第3期466-470,共5页
Progress In Biochemistry and Biophysics
