新型Taq Man-MGB探针在结核分枝杆菌实时PCR检测中的应用

Detection of Mycobacterium Tuberculosis DNA by Real-time Polymerase Chain Reaction using TaqMan-MGB Probe Technology

为建立一种比现有方法敏感、准确性高、重复性好的结核分枝杆菌DNA定性定量检测方法 ,以TaqMan探针技术为基础 ,运用TaqMan MGB探针 ,实时检测临床标本中的结核分枝杆菌DNA .用来自临床标本的DNA及克隆于载体的IS6 1 1 0序列检测所建立方法的有效性 .结果显示 ,所建立方法的最低检测限度为 1个基因拷贝 反应 ,在每反应 1 0 0 ～ 1 0 8拷贝范围内 ,Ct 值同DNA量的对数呈线性关系 .同一模板不同时间或同一时间不同管内扩增 ,所得Ct 值恒定 .用该方法检测 37例结核分枝杆菌培养阳性的痰液标本 ,敏感度为 1 0 0 % ;用该方法检测 1 6例TB系列阴性参考品 ,特异性为1 0 0 % .结果表明 。

To develop a real\|time PCR based on TaqMan technology using the new MGB probe for detecting Mycobacterium tuberculosis DNA, plasmid containing the sequence of interest was constructed. The efficacy of this assay was evaluated by quantitatively measuring levels of the sequence of interest inserted in the plasmid and by detecting the clinical specimens. The conserved regions of Mycobacterium tuberculosis IS6110 element were chosen as primers and MGB probe. Results showed that this assay for Mycobacterium tuberculosis had a detection limit of one genome copy number per reaction. A linear standard curve was obtained between 10\+0 and 10\+8 DNA copies per reaction ( r >0 990). The coefficient of variation for both intra\| and inter\|experimental variability indicated remarkable reproducibility. The sensitivity and specificity of this assay was 100%, respectively. These observations suggested that real\|time PCR based on MGB probe was an excellent candidate for the standard method detecting Mycobacterium tuberculosis.