多特异性内参照模板的构建及应用

Construction of a multispecific internal control for competitive RT-PCR analysis of genes

目的构建多特异性内参照模板,用于Fas、FasL、GB、P、TIA1和βactin的竞争性PCR定量。方法体外合成DNA单链,PCR扩增后,得到2个片段。片段1长145bp,含有Fas、FasL、GB、P、TIA1和βactin的5′引物序列;片段2长147bp,含有相同基因的3′引物序列。将片段1、2分别克隆在载体PKF3上。结果克隆载体经酶切鉴定及DNA序列测定,证实为本实验所设计的多特异性内参照模板,以此为模板,用不同的引物进行PCR扩增,即可得到相应的竞争内参照。应用多特异性内参照模板对移植肾急性排斥患者外周血淋巴细胞中TIA1进行定量,结果显示,底物量在一定范围内,靶序列和竞争性内参照模板的扩增效率基本一致,尤其是在指数期。这一结果同样适用于Fas、FasL、GB、P和βactin的定量。结论本实验构建的多特异性内参照模板可用于6种基因的竞争性PCR检测,为进一步研究打下了基础。

Objective:To construct a mutispecific internal control for competitive RT-PCR analysis of Fas、FasL、GB、P、TIA-1 and β-actin.Methods:Invitro synthesized fragments were amplified by PCR,there are two products,of which 145 and 147 bp.The 145 bp one contained 5′ primer sequences of Fas、FasL、GB、P、TIA-1 and β-actin,another contained 3′ primer sequences of the same genes.The products with restriction sites were inserted into the vector PKF_3.Results:Restriction enzyme analysis and DNA sequencing were used to identify the recombinant plasmid,corresponding internal control was obtained by the amplification of the recombinant plasmid with each primer pair.The mutispecific internal control was then used for quantitative detection of TIA-1 in peripheral blood leukocytes from a patient with acutely rejecting allograft.Our study on Fas、FasL、GB、P、TIA-1 and β-actin showed that the coamplified templates accumulated in a parallel manner throughout not only the exponential phase.Conclusion:The mutispecific internal control can be used for quantitative detection of the six genes. [