TaqMan-MGB荧光定量PCR检测人载脂蛋白A5基因方法的建立及评价

摘要

Objective To establish fluorescence quantitation polymerase chain reaction (PCR) based on TaqMan-MGB probe for detecting apolipoprotein A5 (apoA5) gene. Methods The assay, which was based on specific primers and TaqMan-MGB probe from apoA5 gene, was performed to reconstruct DNA fragment of pPCR-Script-apgA5. The fluorescence quantitation PCR with TaqMan-MGB probe and the standard curve were established. The sensitivity, specificity and repeatability of the TaqMan-MCB probe fluorescence quantitation PCR were determined. Results A fine linear relationship between the threshold cycle (Ct) values of the developed standard curve and the copy number of template was observed (r =0.998). The sensitivity of the assay was 103 copies/μL, the amplification efficiency of assay was 110.2% , and the repeatability was good. Conclusions The fluorescence quantitation PCR based on TaqMan-MGB probe for delecting the apoA5 gene is successfully developed with high sensitivity, specificity and repeatability.%目的 建立以TaqMan-MGB荧光探针为特点的荧光定量聚合酶链反应(PCR),用于检测人载脂蛋白A5(apo A5)基因.方法 针对人apo A5基因设计特异性引物和TaqMan-MGB荧光探针,以重组克隆质粒pPCR-Script-apo A5为DNA模板,在荧光定量PCR仪上建立TaqMan-MGB荧光定量PCR检测方法和标准曲线,进行灵敏度、重复性、特异性实验.结果 建立的定量标准曲线阈值循环数(Ct)与模板拷贝数呈良好线性关系(r=0.998)；最低检测浓度为103拷贝/μL；扩增效率(E)为110.2％,且重复性好.结论 成功建立检测人apoA5基因的TaqMan-MGB荧光定量PCR.该法具有较好的灵敏度、特异性及重复性.