全自动(定量PCR)基因扩增诊断仪配套定量试剂的研制

A quantitative PCR kit used in automatic genetic amplification instrument

目的 研制一种能用于仪器操作的灵敏、快速、特异的定量试剂 .方法 使用一个生物素标记的上游引物 ,对HBVDNA及其竞争模板DNA进行不对称PCR扩增 ,使扩增出的单链带有生物素 ,并可结合在固定有链亲合素的微孔板的表面 .用标有辣根过氧化物酶的待测模板探针和竞争模板探针分别与两个微孔中固定的相应单链PCR产物杂交 ,而后加底物显色 ,测定吸光度进行分级定量分析 .结果 该定量方法可以检测 10拷贝数的模板 ,而且对简便处理的标本适应性强 ;以分级定量的形式定量时 ,具有良好的重复性 .结论 该试剂适用于仪器操作 。

AIM A sensitive, rapid and specific quantitative reagent used in quantitative PCR instrument was researched and the content of HBV DNA in serum was quantified.METHODS The HBV DNA and the competitive DNA template were asymmetrically amplified by a bio labeled upstream primer, the single string PCR products with Biotin were combined to the surface of the streptavidin fixed wells. Then the HRP labeled sample probe and the competitive probe were hybridized with the single string PCR products fixed in two wells respectively, and then the substrate was added to and Ab values were examined. The result of quantitative assay was given by computer. RESULTS 10 copy template can be quantified by this kit. The simple preparation of samples DNA is also adapted to this kit and the results can be repeated. CONCLUSION This kit can be used in instrument and quantify the content of HBV DNA in serum.